# Patent

## Title
High-throughput screen for identifying selective persistent sodium channels channel blockers

## Abstract

A method for identifying a selective persistent Na+ channel blocker by measuring the ability of the blocker to reduce or inhibit a persistent Na+ current to a greater degree than a transient Na+ current. Aspects of the present method provide Na+ depletion/repletion methods for identifying a selective blocker of a persistent Na+ channel, hyperpolarization methods for identifying a blocker of a persistent Na+ channel, and Na/K ATPase pump inhibitor methods for identifying a selective blocker of a persistent Na+ channel.

## Background
This application is a continuation-in-part and claims priority pursuant to 35 U.S.C. § 120 to U.S. Pat. No. 6,991,910 application Ser. No. 09/989,797, filed Nov. 20, 2001, which is hereby incorporated by reference in its entirety.


All of the patents and publications cited in this application are hereby incorporated by reference in their entirety.
The lipid bilayer membrane of all cells forms a barrier that is largely impermeable to the flux of ions and water. Residing within the membrane is a superfamily of proteins called ion channels, which provide selective pathways for ion flux. Precisely regulated conductances produced by ion channels are required for intercellular signaling and neuronal excitability. In particular, a group of ion channels that open upon depolarization of excitable cells are classified as voltage-gated and are responsible for electrical activity in nerve, muscle and cardiac tissue. In neurons, ion currents flowing through voltage-gated sodium ion (Na+) channels are responsible for rapid spike-like action potentials. During action potentials the majority of Na+ channels open very briefly. These brief openings result in transient Na+ currents. However, a subset of voltage-gated Na+ channels does not close rapidly, but remain open for relatively long intervals. These channels therefore generate sustained or persistent Na+ currents. The balance between transient and persistent Na+ current is crucial for maintaining normal physiological function and electrical signaling throughout the entire nervous system.
Over the past 50 years, an increasing number of diseases of the nervous system and other excitable tissues have been shown to result from the dysregulation of ion channels. This class of disease has been termed channelopathies. Aberrant persistent sodium current can contribute to the development or progression of many channelopathic conditions because normal function is disrupted when neurons discharge signals inappropriately. For example, abnormal persistent sodium current is thought to induce deleterious phenomena, including, e.g., neuropathies, neurodegenerative diseases, movement disorders, cardiac arrhythmia, epileptic seizure, neuronal cell death, behavioral disorders and dementia, see, e.g., Robert S. Kass, The Channelopathies: Novel Insights into Molecular and Genetic Mechanisms of Human Disease, 115(8) J. Clin. Invest. 1986-1989 (2005); Alfred L. George, Inherited Disorders of Voltage-gated Sodium Channels, 115(8) J. Clin. Invest. 1990-1999 (2005); Karin Jurkat-Rott and Frank Lehmann-Horn, Muscle Channelopathies and Critical Points in Functional and Genetic Studies, 115(8) J. Clin. Invest. 2000-2009 (2005); Miriam H. Meisler and Jennifer A. Kearney, Sodium Channel Mutations in Epilepsy and Other Neurological Disorders, 115(8) J. Clin. Invest. 2010-2017 (2005); Arthur J. Moss and Robert S. Kass, Long QT Syndrome: from Channels to Cardiac Arrhythmias, 115(8) J. Clin. Invest. 2018-2024 (2005); Christoph Lossin et al., Molecular Basis of an Inherited Epilepsy 34(6) NEURON 877-84 (2002); Peter K. Stys et al., Ionic Mechanisms of Anoxic Injury in Mammalian CNS White Matter: Role of Na+ Channels and Na(+)—Ca2+ Exchanger, 12(2) J. NEUROSCI. 430-439 (1992); Peter K. Stys et al., Noninactivating, Tetrodotoxin-Sensitive Na+ Conductance in Rat Optic Nerve Axons, 90(15) PROC. NATL. ACAD. SCI. USA, 6976-6980 (1993); and Giti Garthwaite et al., Mechanisms of Ischaemic Damage to Central White Matter Axons: A Quantitative Histological Analysis Using Rat Optic Nerve, 94(4) NEUROSCIENCE 1219-1230 (1999). For example, in the case of the neuropathies embraced by epilepsy, there can be a brief electrical “storm” arising from neurons that are inherently unstable because of a genetic defect as in various types of inherited epilepsy, or from neurons made unstable by metabolic abnormalities such as low blood glucose, or alcohol. In other cases, the abnormal discharge can come from a localized area of the brain, such as in patients with epilepsy caused by head injury or brain tumor. In the case of ischemic injuries, such as, e.g., cerebral ischemia and myocardial ischemia, there can be prolonged electrical activity arising from neurons in which persistent sodium channel expression or activity is increased. Such aberrant electrical activity can cause or contribute to neuronal death, which can lead to debilitating injury or death of an individual. Aberrant electrical activity also can contribute to neurodegenerative disorders such as, without limitation, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis. Thus, aberrant persistent sodium current can contribute to development or progression of pathological conditions by collapsing the normal cell transmembrane gradient for sodium, leading to reverse operation of the sodium-calcium exchanger, and resulting in an influx of intracellular calcium, which injures the axon, see, e.g., Stys et al., supra, (1992). Therefore, selective reduction in the expression or activity of sodium channels capable of mediating persistent current relative to any reduction in normal voltage-gated (transient) sodium current can be useful for treating channelopathic conditions associated with increased persistent sodium current.
Recent evidence has revealed that increased activity from persistent Na+ channels may be responsible for the underlying basis of chronic pain, see e.g., Fernando Cervero & Jennifer M. A. Laird, Role of Ion Channels in Mechanisms Controlling Gastrointestinal Pain Pathways, 3(6) CURR. OPIN. PHARMACOL. 608-612 (2003); Joel A. Black et al., Changes in the Expression of Tetrodotoxin-Sensitive Sodium Channels Within Dorsal Root Ganglia Neurons in Inflammatory Pain, 108(3) PAIN 237-247 (2004) and Li Yunru et al., Role of Persistent Sodium and Calcium Currents in Motoneuron Firing and Spasticity in Chronic Spinal Rats, 91(2) J. NEUROPHYSIOL. 767-783 (2004). Alterations in persistent sodium channel expression and/or function has a profound effect on the firing pattern of neurons in both the peripheral and central nervous systems. For example, injury to sensory primary afferent neurons often results in rapid redistribution of persistent sodium channels along the axon and dendrites and in abnormal, repetitive discharges or exaggerated responses to subsequent sensory stimuli. Such an exaggerated response is considered to be crucial for the incidence of spontaneous pain in the absence of external stimuli that is characteristic of chronic pain. In addition, inflammatory pain is associated with lowered thresholds of activation of nociceptive neurons in the periphery and altered persistent sodium channel function is thought to underlie aspects of this phenomenon. Likewise, neuropathic pain states resulting from peripheral nerve damage is associated with altered persistent sodium channel activity and ectopic action potential propagation. Therefore, selective reduction in the expression or activity of sodium channels capable of mediating persistent current relative to any reduction in normal voltage-gated (transient) sodium current can be useful for treating chronic pain conditions associated with increased persistent sodium current.
Besides their importance under physiological conditions, Na+ channels are also important under pathophysiological situations. For example they appear play a role in epileptic seizures, cardiac arrhythmias, and ischemia/hypoxia-induced cardiac and neuronal cell death (Taylor et al, 1997; Ragsdale et al, 1998). Importantly, the persistent Na+ current appears to play a major role in generating the above mentioned cellular abnormalities (Stys, 1998; Taylor et al, 1997). For example persistent Na+ current is unregulated in both cardiac and neuronal cells during hypoxia (Saint et al, 1996; Hammarstrom, 1998) and may ultimately lead to overload of cell Na+ and calcium, conditions leading to cell death (Stys, 1998). Blockers of voltage-gated Na+ channels have been shown to be effective in ameliorating cellular dysfunctions and death resulting from errant operation of voltage-gated sodium channels (Stys, 1998). However, in many cases these blockers inhibit both the normal inactivating (transient) and non-inactivating (persistent) Na+ channels to the same extent. Significant block of normal transient Na+ channels could seriously compromise cellular and organ function or may even cause death. Thus assuming that the persistent Na+ current is the therapeutic target, it is important to develop drugs that will block this component of Na+ current but not the normal transient. However, in order to discern whether a compound selectively blocks the persistent over the transient Na+ current conventional electrophysiological methods such as whole cell patch clamping or voltage clamping in oocyte preparations must be performed (Marty and Neher, 1995; Shih et al, 1998).
Thus, there exists a need for new screening methods that can be used to identify persistent Na+ channel blockers useful for treating channelopathies and chronic pain. The present invention satisfies this need and provides related advantages as well, such as, e.g., high-throughput screens for identifying voltage-gated Na+ channel blockers that selectively reduce or prevent persistent Na+ currents.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 a schematic of a basic ion channel mechanism. The top illustration shows a K+ channel and K+ ion flow, a transient Na+ channel and ion flow, a persistent Na+ channel and ion flow and a Na/K ATPase pump and K+ and Na+ ion flow. While drawn as separate channels here, the same Na+ channel can have both transient and persistent current properties. The bottom panels show a current recording from a cell containing a transient Na+ current (left panel) and a recording from a cell containing both transient and persistent Na+ currents (right panel). Current vs. time is plotted for a voltage-gated Na+ current. In the left panel, the initial transient current is shown during a 2 msec depolarization. In the right panel, the initial transient current and a smaller sustained persistent current are shown during a 200 msec depolarization.
FIG. 2 shows a schematic of a Na+ depletion/repletion protocol. Four steps integral to the assay are illustrated. 1) Providing Sample: A cell containing the principal components of the assay—a K+ channel and channel(s) capable of producing transient and persistent Na+ currents are incubated in Na+-free solution containing a voltage-sensitive dye (Dye) and a test compound (Blocker). 2) Depolarizing the cell: A small aliquot of solution containing concentrated K+ is added to the solution to initiate a depolarization of the membrane sufficient to activate the Na+ channels. In the absence of external Na+ to act as charge carrier through the Na+ channels only small background K+-induced depolarization and fluorescence change is produced (see also FIG. 3). 3) Generating current and detecting fluorescence emitted in the absence of an effective persistent Na+ channel blocker: Following and interval sufficient to allow the closure of transient Na+ channels, an aliquot of solution containing concentrated Na+ sufficient to raise the external Na+ concentration to physiological levels is added. In the absence of an effective persistent Na+ channel blocker, Na+ ions acting as a charge carrier through persistent Na+ channels produce a depolarization of the cell membrane and a subsequent change in fluorescence of the voltage-sensitive dye. 4) Generating current and detecting fluorescence emitted in the presence of an effective persistent Na+ channel blocker: With solution additions as in (3) above, except that the solution now contains an effective blocker of persistent Na+ channels, Na+ ions are prevented from entering the cell, no depolarization occurs and no change in fluorescence is observed.
FIG. 3 shows a graphic depiction of an emitted fluorescence readout using a Na+ depletion/repletion protocol. Fluorescence in relative fluorescence units vs. time is plotted for a voltage-gated Na+ current. The recording of a molecule exhibiting a blocking activity of a persistent Na+ current is indicated by a black line. A control sample which lacks blocking activity of a persistent Na+ current is indicated by a dashed line.
FIG. 4 shows a schematic of a hyperpolarization protocol. The hyperpolarization protocol illustrated here is similar to the depletion/repletion protocol shown in FIG. 2, except that test cell is engineered to have approximately equal K+ and Na+ conductances and a resting potential midway between the equilibrium potentials for K+ and Na+. This resting potential would also be engineered to result in the inactivation of transient Na+ channels. Three steps integral to the assay are illustrated. 1) Providing Sample: A cell containing the principal components of the assay—a K+ channel and channel capable of producing persistent Na+ currents are incubated in Na+-containing solution which also contains a voltage-sensitive dye (Dye). In this case Na+ ions acting as charge carriers through the open persistent Na+ channels will result in a steady-state depolarization of the cell membrane and significant fluorescent emission from the voltage-sensitive dye. 2) Adding a potential blocker (ineffective compound) and detecting fluorescence emitted: If the compound is ineffective in blocking the persistent Na+ current, no change in Na+ influx, depolarization or emitted fluorescence will occur. 3) Adding a potential blocker (effective compound) and detecting fluorescence emitted: If the compound is effective in blocking the persistent Na+ current, a decrease in Na+ influx, depolarization and emitted fluorescence will occur.
FIG. 5 shows a graphic depiction of an emitted fluorescence readout using a hyperpolarization protocol. Fluorescence in relative fluorescence units vs. time is plotted for a voltage-gated Na+ current. The recording of a molecule exhibiting a blocking activity of a persistent Na+ current is indicated by a black line. A control sample which lacks blocking activity of a persistent Na+ current is indicated by a dashed line.
FIG. 6 shows a schematic of a Na/K pump inhibitor protocol. Two steps integral to the assay are illustrated. 1) Adding Ouabain: In a Cl-free physiological solution ouabain (100 uM) is added to engineered cells where GK is at least 20 fold>than GNa+ persistent. 2) Detecting fluorescence emitted: Inhibition of the Na/K pump causes the cells to exchange extracellular Na+ (via influx through persistent Na+ channels) for intracellular K. The loss of K changes EK favoring cell depolarization, reflected as an increase in fluorescence intensity for the case of an anionic dye. In the presence of a persistent Na+ channel blocker the K-dependent depolarization will be inhibited.
FIG. 7 shows a graphic depiction of an emitted fluorescence readout using a Na/K pump inhibitor protocol. Fluorescence in relative fluorescence units vs. time is plotted for a voltage-gated Na+ current. The recording of a molecule exhibiting a blocking activity of a persistent Na+ current is indicated by a black line. A control sample which lacks blocking activity of a persistent Na+ current is indicated by a dashed line.
FIG. 8 shows a schematic of a transient blocker protocol. 1) Field stimulation of cells in wells: The engineered cells are placed in wells containing an appropriate physiological solution and a pair of stimulating electrodes capable of passing sufficient current to reach threshold for action potential initiation. 2) Detection of emitted fluorescence following stimulation to threshold: Depolarization of the cell following the upstroke to the action potential is detected by an increase in emitted fluorescence. 3) Adding a potential blocker and detecting emitted fluorescence. If a compound is a persistent Na+ channel blocker an increase in emitted fluorescence will not be detected.
FIG. 9 shows a graphic depiction of an emitted fluorescence readout using a transient blocker protocol. Fluorescence in relative fluorescence units vs. time is plotted for a voltage-gated Na+ current. The recording of a molecule exhibiting a blocking activity of a transient Na+ current is indicated by a black line. A control sample which lacks blocking activity of a transient Na+ current is indicated by a dashed line. A selective persistent Na+ channel blocker would lack significant blocking activity of a transient Na+ current and thus behave more like a control sample.
FIG. 10 shows inhibition of persistent current-dependent depolarization by Na+ channel blockers. In this assay, cells are resting in wells containing a 80 μL solution of 140 mM TEA-MeSO3 (Na+-free box) to which is added 240 μL solution of 140 mM NaMeSO3 and 13 mM KMeSO3 for a final K+ concentration of 10 mM and a final Na+ concentration of 110 mM (Na+/K+-addition). This elicits a robust depolarizing response. Following the resolution of the sodium-dependent depolarization, a second aliquot of KMeSO3 is added to the well bringing the final K+ concentration to 80 mM (high potassium-addition). This addition results in a second depolarizing response. Compounds that reduce the sodium-dependent, but not the potassium-dependent depolarizations are selected as persistent sodium channel blockers. Circles indicate the control response with 0.1% DMSO added, triangles show the effects of the sodium channel inhibitor tetracaine (10 μM) and the diamonds show the response during the application of a non-specific channel blocker.
FIG. 11 shows data from assays in which the screening window for the persistent current assay is determined. To evaluate the size of the “screening window,” data was examined from assays in which responses to sodium-dependent depolarization were measured in the presence of 10 μM Tetracaine to completely block the sodium-dependent depolarization or in the presence of a 0.1% DMSO control to obtain a maximum depolarization. Data were binned into histograms and a screening window (Z) was calculated from the mean and standard deviation for the maximal and minimum values according to the equation: Z=1−(3×STDMax+3×STDMin)/(MeanMax−MeanMin). Histograms A, B and C represent data obtained from three different assay plates. The screening window for a run was considered adequate 1.0≧Z≧0.5.
FIG. 12 shows sodium current traces before and after the addition of 3 μM Compound 1 or 500 nM TTX. HEK cells expressing Nav 1.3 channels were patch clamped in the perforated-patch mode. Currents were elicited by 200 msec test pulses to 0 mV from a holding potential of −90 mV.
FIG. 13 shows a dose-response curve for Compound 1. The peak amplitudes of transient Na+ current (It) and the steady state amplitude of the persistent current (Ip) were measured at various Compound 1 concentrations, normalized to the amplitude of the control currents. The percent block was then plotted against drug concentration. Solid lines represent fits to the data with the Hill equation. The calculated IC50 values and Hill coefficients are as follows: Hill slope, It is 0.354 and Ip is 0.733; IC50, It is 0.167 M and Ip is 3.71×10−6 M.
FIG. 14 shows the use of a single-wavelength dye to measure persistent Na+ channel activity to measure fluorescence on the FLIPR-Tetra. (A) Raw fluorescence signals are shown during application of the Na+ depletion/repletion protocol. Base line measurements in the presence of a Na+-free buffer are shown for the first 4 seconds of the record. After establishing of the base line fluorescence, a depolarizing buffer containing Na+ is applied to the well resulting a an initial rapid increase in fluorescence followed by a longer sustained increase (Control). In the presence of a saturating concentration of TTX (1 μM TTX) to block all the Na+ channel mediated signal, the initial response is lost and only the sustained non-channel mediated response remains. (B) Subtraction of the TTX resistant response from the control response reveals the persistent Na+ current mediated signal.
FIG. 15 shows typical compound plate layouts for the persistent current assay on the FLIPR-tetra. (A) In the screening window format a 96-well compound plate is organized such that columns 1-5 & 8-12 contain the normal Na+ repletion solution while columns 6-7 contain the Na+ repletion solution plus 1 μM TTX. (B) For the drug screening dose-response protocol columns 1-2 & 11-12 were used for positive and negative controls. The remaining columns contained different concentrations of the test compound with the highest concentration in column 10 and serial three-fold dilutions from column 9 through 3.
FIG. 16 shows the results of the screening window experiment. (A) A screenshot from the FLIPR-Tetra. Plate was loaded as described in FIG. 15 A. Data are presented following the subtraction of the non-specific (TTX-resistant) response. (B) Measurement of the peak response in the presence and absence of 1 μM TTX. Data are displayed as mean±SD. Z′ factor of 0.70 was calculated as described in example 2 demonstrates an acceptable screening window for this assay.
FIG. 17 shows a dose response analysis using the FLIPR-tetra based persistent current assay. (A) Illustrates the persistent Na+ current mediated signal in wells loaded as described in FIG. 15 B. Columns 1 and 11 illustrate a negative control with no persistent current blocker and columns 2 and 12 show positive controls in the presence of 1 μM TTX. The remaining columns show a dose-response for TTX with increasing concentrations left to right. (B) The averaged responses from each column are plotted vs. the time for the TTX dose response. (C) Averaged data for TTX, Lidocaine, and Tetracaine are plotted as a semi-log dose response as mean±SD, the data is fitted by logistic function (lines) and the estimated IC50 values are shown (mean±SD).
FIG. 18 shows transient Na+ currents taken from 4 wells on an IonWorks automated patch clamp device. Control (Pre-addition) and TTX blocked (100 nM TTX) traces are shown for each cell.
FIG. 19 shows a dose response analysis of the IonWorks transient current assay. Averaged currents are plotted as a semi-log dose response as mean±SD in the presence of increasing concentrations of TTX. The data is fitted by logistic function (line) and the estimated IC50 value is shown.



DETAILED DESCRIPTION
In the normal functioning of the nervous system, neurons are capable of receiving a stimulus, and in response, propagating an electrical signal away from their neuron cell bodies (soma) along processes (axons). From the axon, the signal is delivered to the synaptic terminal, where it is transferred to an adjacent neuron or other cell. It is the action potential that is responsible for electrical transmission in the nervous system, and contractility in the heart and skeletal muscle, see, e.g., Bertil Hille, Ion Channels of Excitable Membranes 3rd ed. Sinauer Associates, Inc. (Sunderland, Mass.) 2001. Generally, under resting conditions, sodium channels are closed until a threshold stimulus depolarizes the cell membrane. During membrane depolarization, sodium channels activate by opening the channel pore briefly (one millisecond) to rapidly generate the upstroke of the action potential and then inactivate by closing the channel pore until the excitable cell returns to its resting potential and the sodium channels re-enter the resting state.
Without wishing to be bound by the following, this behavior of voltage-gated sodium channels can be understood as follows. Sodium channels can reside in three major conformations or states. The resting or closed state predominates at membrane potentials more negative than approximately −60 mV. Upon depolarization, the channels open rapidly to allow current flow and, thereby, enter the active state. The transition from resting to active states occurs within a millisecond after depolarization to positive membrane potentials. Finally during sustained depolarizations of greater than 1-2 ms, the channels enter a second closed or inactivated state. Subsequent re-openings of the channels require a recycling of the channels from the inactive to the resting state, which occurs when the membrane potential returns to negative values. This means that membrane depolarization not only opens sodium channels but also causes them to close even during sustained depolarizations (Hodgkin and Huxley, 1952). Thus normal Na+ channels open briefly during depolarization and are closed at rest.
However, some Na+ channels may be open under resting conditions at relatively negative membrane potentials and even during sustained depolarization (Stys, 1998; Taylor, 1993). These non-inactivating Na+ channels generate what is known as a persistent Na+ current, see FIG. 1. Persistent Na+ channels have these properties because they activate (open) at more negative membrane potentials than normal Na+ channels and inactivate at more positive potentials (Alonso et al, 1999). This means that these persistent Na+ channels may be open at membrane potentials as negative as −80 mV (Stys, 1998) and stay open at potentials as positive as 0 mV (Alonso, et al, 1999). These persistent Na+ channels are thought to be involved in synaptic amplification and modification of spiking behavior and also in the generation of conditions leading to cellular dysfunction (Ragsdale et al, 1998; and Taylor, 1993). This unique property of persistent Na+ channels is exploited in the assays in accordance with the present invention.
Aspects of the present invention provide Na+ depletion/repletion methods for identifying a selective blocker of a persistent Na+ channel, such method comprising the steps of a) providing a test sample 1 comprising a Na+-free physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; and a potential Na+ channel blocker; b) depolarizing the membrane of the cell in the test sample 1; c) generating a current through the persistent Na+ channel by adding Na+ to test sample 1 at least 10 msec after step (b); d) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1; e) providing a control sample 1 comprising a Na+-free physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; f) depolarizing the membrane of the cell in the control sample 1; g) generating a current through the persistent Na+ channel by adding Na+ ions to the control sample 1 at least 10 msec after step (f); h) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1; i) comparing the emitted fluorescence from step (d) to the emitted fluorescence from step (h).
Other aspects of the present invention Na+ depletion/repletion provide a method for identifying a selective blocker of a persistent Na+ channel, such method comprising the steps of a) providing a test sample 1 comprising a Na+-free physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel, and a potential Na+ channel blocker; b) depolarizing the membrane of the cell in the test sample 1; c) generating a current through the persistent Na+ channel by adding Na+ to test sample 1 at least 10 msec after step (b); d) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1; e) providing a control sample 1 comprising a Na+-free physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; f) depolarizing the membrane of the cell in the control sample 1; g) generating a current through the persistent Na+ channel by adding Na+ ions to the control sample 1 at least 10 msec after step (f); h) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1; i) determining the relative emitted fluorescence 1 by comparing the emitted fluorescence from step (d) to the emitted fluorescence from step (h); j) providing a test sample 2 comprising a physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel and a transient Na+ channel, and a potential Na+ channel blocker; k) depolarizing membrane of the cell in test sample 2; l) detecting the fluorescence emitted by the voltage-sensitive dye in test sample 2; m) providing a control sample 2 comprising a physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a transient Na+ channel; n) depolarizing membrane of the cell in control sample 2; o) detecting the fluorescence emitted by the voltage-sensitive dye in control sample 2; p) determining a relative emitted fluorescence 2 by comparing the emitted fluorescence from step (l) to the emitted fluorescence from step (o); and q) comparing the relative emitted fluorescence 1 in step (i) with the relative emitted fluorescence 2 in step (p).
Other aspects of the present invention provide a hyperpolarization method for identifying a blocker of a persistent Na+ channel, such method comprising the steps of a) providing a test sample 1 comprising a physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a persistent Na+ channel wherein a resting membrane potential of the cell is approximately halfway between an equilibrium potential of Na+ and an equilibrium potential of K+; b) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1; c) adding a potential Na+ channel blocker to test sample 1; d) detecting fluorescence emitted by the voltage-sensitive dye in the test sample 1; e) comparing the emitted fluorescence from step (b) with the emitted fluorescence from step (d).
Other aspects of the present invention provide a hyperpolarization method for identifying a blocker of a persistent Na+ channel, such method comprising the steps of a) providing a test sample 1 comprising a physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a persistent Na+ channel wherein a resting membrane potential of the cell is approximately halfway between an equilibrium potential of Na+ and an equilibrium potential of K+; b) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1; c) adding a potential Na+ channel blocker to test sample 1; d) detecting fluorescence emitted by the voltage-sensitive dye in the test sample 1; e) determining a relative emitted fluorescence 1 by comparing the emitted fluorescence from step (b) with the emitted fluorescence from step (d); f) providing a test sample 2 comprising a physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel and a transient Na+ channel, and a potential Na+ channel blocker; g) depolarizing the membrane of the cell in test sample 2; h) detecting the fluorescence emitted by the voltage-sensitive dye in test sample 2; i) providing a control sample 2 comprising a physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a transient Na+ channel; j) depolarizing the membrane of the cell in control sample 2; k) detecting the fluorescence emitted by the voltage-sensitive dye in control sample 2; l) determining a relative emitted fluorescence 2 by comparing the emitted fluorescence from step (h) relative to an emitted fluorescence from step (k); and m) comparing the relative emitted fluorescence in step (e) with the relative emitted fluorescence in step (l).
Other aspects of the present invention provide a Na/K ATPase pump inhibitor method for identifying a blocker of a persistent Na+ channel, such method comprising the steps of a) providing a test sample 1 comprising a Cl−-free physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel and a persistent Na+ channel wherein a K+ conductance of the K+ channel is at least 20-fold higher than a Na+ conductance from the persistent Na+ channel, and a potential Na+ channel blocker; b) depolarizing the membrane of the cell with a Na/K pump inhibitor to the test sample 1; c) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1; d) providing a control sample 1 comprising a Cl−-free physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a persistent Na+ channel wherein a K+ conductance of the K+ channel is at least 20-fold higher than a Na+ conductance from the persistent Na+ channel; e) depolarizing the membrane of the cell with a Na/K pump inhibitor to the control sample 1; f) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1; g) comparing the emitted fluorescence from step (c) to the emitted fluorescence from step (f).
Other aspects of the present invention provide a Na/K ATPase pump inhibitor method for identifying a selective blocker of a persistent Na+ channel, such method comprising the steps of a) providing a test sample 1 comprising a Cl−-free physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel and a persistent Na+ channel wherein a K+ conductance of the K+ channel is at least 20-fold higher than a Na+ conductance from the persistent Na+ channel, and a potential Na+ channel blocker; b) depolarizing the membrane of the cell with a Na/K pump blocker to the test sample 1; c) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1; d) providing a control sample 1 comprising a Cl−-free physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a persistent Na+ channel wherein a K+ conductance of the K+ channel is at least 20-fold higher than a Na+ conductance from the persistent Na+ channel; e) depolarizing the membrane of the cell with a Na/K pump blocker to the control sample 1; f) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1; g) comparing the emitted fluorescence from step (c) to the emitted fluorescence from step (f); h) providing a test sample 2 comprising a physiological solution, a voltage-sensitive fluorescence dye, a cell having a K+ channel and a transient Na+ channel, and a potential Na+ channel blocker; i) depolarizing the membrane of the cell in test sample 2; j) detecting the fluorescence emitted by the voltage-sensitive dye in test sample 2; k) providing a control sample 2 comprising a physiological solution, a voltage-sensitive fluorescence dye, and a cell having a K+ channel and a transient Na+ channel; l) depolarizing the membrane of the cell in control sample 2; m) detecting the fluorescence emitted by the voltage-sensitive dye in control sample 2; n) comparing the emitted fluorescence from step (j) relative to an emitted fluorescence from step (m); and o) comparing the difference in step (g) with the difference in step (n).
Aspects of the present invention provide, in part, a selective persistent Na+ current blocker. As used herein, the term “persistent Na+ current blocker” means any molecule that for at least one particular dose can reduce or prevent a persistent Na+ current. As used herein, the term “selective persistent Na+ current blocker” means any molecule that for at least one particular dose can selectively reduce or prevent a persistent Na+ current as compared to a transient Na+ current. As used herein, the term “selective” means to have a unique effect or influence or reacting in only one way or with only one thing. It is envisioned that a selective persistent Na+ channel blocker can modulate a persistent Na+ current derived from at least one persistent Na+ channel in an antagonistic manner by reducing or preventing a persistent Na+ current. It is further envisioned that a selective persistent Na+ channel blocker acting in an antagonistic manner can do so in a competitive or non-competitive way. Non-limiting examples of a selective persistent Na+ channel blocker acting in an antagonistic manner include, e.g., a persistent Na+ channel pan-antagonist that reduces or prevents persistent Na+ current generated from all persistent Na+ channel subunits, a persistent Na+ channel-selective antagonist that reduces or prevents persistent Na+ current generated from a subgroup of persistent Na+ channel subunits, and a persistent Na+ channel-specific antagonist that reduces or prevents persistent Na+ current generated from only one persistent Na+ channel subunit.
In an aspect of this embodiment, a selective persistent Na+ current blocker prevents persistent Na+ current but does not affect a transient Na+ current. In aspects of this embodiment, a selective persistent Na+ current blocker prevents persistent Na+ current and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current. In other aspects of this embodiment, a selective persistent Na+ current blocker reduces a persistent Na+ current by, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%, and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current.
In aspects of this embodiment, a selective persistent Na+ current blocker can reduce a persistent Na+ current by, e.g., at least 20-fold more than transient sodium current is reduced, at least 30-fold more than transient sodium current is reduced, at least 40-fold more than transient sodium current is reduced, at least 50-fold more than transient sodium current is reduced, at least 60-fold more than transient sodium current is reduced, at least 70-fold more than transient sodium current is reduced, at least 80-fold more than transient sodium current is reduced, at least 90-fold more than transient sodium current is reduced or at least 100-fold more than transient sodium current is reduced. In yet other aspects of this embodiment, a selective persistent Na+ current blocker can reduce a persistent Na+ current by, e.g., at least 100-fold more than transient sodium current is reduced, at least 200-fold more than transient sodium current is reduced, at least 300-fold more than transient sodium current is reduced, at least 400-fold more than transient sodium current is reduced, at least 500-fold more than transient sodium current is reduced, at least 600-fold more than transient sodium current is reduced, at least 700-fold more than transient sodium current is reduced, at least 800-fold more than transient sodium current is reduced, at least 900-fold more than transient sodium current is reduced or at least 1000-fold more than transient sodium current is reduced.
In an embodiment, selective persistent Na+ current blocker can be a persistent Na+ channel pan-antagonist. In an aspect of this embodiment, a persistent Na+ channel pan-antagonist prevents persistent Na+ current generated from all persistent Na+ channel subunits but does not affect a transient Na+ current. In aspects of this embodiment, a persistent Na+ channel pan-antagonist prevents persistent Na+ current generated from all persistent Na+ channel subunits and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current. In other aspects of this embodiment, a persistent Na+ channel pan-antagonist reduces persistent Na+ current generated from all persistent Na+ channel subunits by, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%, and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current.
In aspects of this embodiment, a persistent Na+ channel pan-antagonist can reduce persistent Na+ current generated from all persistent Na+ channel subunits by, e.g., at least 20-fold more than transient sodium current is reduced, at least 30-fold more than transient sodium current is reduced, at least 40-fold more than transient sodium current is reduced, at least 50-fold more than transient sodium current is reduced, at least 60-fold more than transient sodium current is reduced, at least 70-fold more than transient sodium current is reduced, at least 80-fold more than transient sodium current is reduced, at least 90-fold more than transient sodium current is reduced or at least 100-fold more than transient sodium current is reduced. In yet other aspects of this embodiment, a persistent Na+ channel pan-antagonist can reduce a persistent Na+ current generated from all persistent Na+ channel subunits by, e.g., at least 100-fold more than transient sodium current is reduced, at least 200-fold more than transient sodium current is reduced, at least 300-fold more than transient sodium current is reduced, at least 400-fold more than transient sodium current is reduced, at least 500-fold more than transient sodium current is reduced, at least 600-fold more than transient sodium current is reduced, at least 700-fold more than transient sodium current is reduced, at least 800-fold more than transient sodium current is reduced, at least 900-fold more than transient sodium current is reduced or at least 1000-fold more than transient sodium current is reduced.
In an embodiment, selective persistent Na+ current blocker can be a persistent Na+ channel-selective antagonist. In an aspect of this embodiment, a persistent Na+ channel-selective antagonist prevents persistent Na+ current generated from a subgroup of persistent Na+ channel subunits but does not affect a transient Na+ current. In aspects of this embodiment, a persistent Na+ channel-selective antagonist prevents persistent Na+ current generated from a subgroup of persistent Na+ channel subunits and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current. In other aspects of this embodiment, a persistent Na+ channel-selective antagonist reduces persistent Na+ current generated from a subgroup of persistent Na+ channel subunits by, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%, and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current.
In aspects of this embodiment, a persistent Na+ channel-selective antagonist can reduce persistent Na+ current generated from a subgroup of persistent Na+ channel subunits by, e.g., at least 20-fold more than transient sodium current is reduced, at least 30-fold more than transient sodium current is reduced, at least 40-fold more than transient sodium current is reduced, at least 50-fold more than transient sodium current is reduced, at least 60-fold more than transient sodium current is reduced, at least 70-fold more than transient sodium current is reduced, at least 80-fold more than transient sodium current is reduced, at least 90-fold more than transient sodium current is reduced or at least 100-fold more than transient sodium current is reduced. In yet other aspects of this embodiment, a persistent Na+ channel-selective antagonist can reduce a persistent Na+ current generated from a subgroup of persistent Na+ channel subunits by, e.g., at least 100-fold more than transient sodium current is reduced, at least 200-fold more than transient sodium current is reduced, at least 300-fold more than transient sodium current is reduced, at least 400-fold more than transient sodium current is reduced, at least 500-fold more than transient sodium current is reduced, at least 600-fold more than transient sodium current is reduced, at least 700-fold more than transient sodium current is reduced, at least 800-fold more than transient sodium current is reduced, at least 900-fold more than transient sodium current is reduced or at least 1000-fold more than transient sodium current is reduced.
In an embodiment, selective persistent Na+ current blocker can be a persistent Na+ channel-specific antagonist. In an aspect of this embodiment, a persistent Na+ channel-specific antagonist prevents persistent Na+ current generated from only one persistent Na+ channel subunit but does not affect a transient Na+ current. In aspects of this embodiment, a persistent Na+ channel-specific antagonist prevents persistent Na+ current generated from only one persistent Na+ channel subunits and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current. In other aspects of this embodiment, a persistent Na+ channel-specific antagonist reduces persistent Na+ current generated from only one persistent Na+ channel subunits by, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%, and affects, e.g., at most 5% of a transient Na+ current, at most 10% of a transient Na+ current, at most 15% of a transient Na+ current, at most 20% of a transient Na+ current or at most 25% of a transient Na+ current.
In aspects of this embodiment, a persistent Na+ channel-specific antagonist can reduce persistent Na+ current generated from only one persistent Na+ channel subunits by, e.g., at least 20-fold more than transient sodium current is reduced, at least 30-fold more than transient sodium current is reduced, at least 40-fold more than transient sodium current is reduced, at least 50-fold more than transient sodium current is reduced, at least 60-fold more than transient sodium current is reduced, at least 70-fold more than transient sodium current is reduced, at least 80-fold more than transient sodium current is reduced, at least 90-fold more than transient sodium current is reduced or at least 100-fold more than transient sodium current is reduced. In yet other aspects of this embodiment, a persistent Na+ channel-specific antagonist can reduce a persistent Na+ current generated from only one persistent Na+ channel subunits by, e.g., at least 100-fold more than transient sodium current is reduced, at least 200-fold more than transient sodium current is reduced, at least 300-fold more than transient sodium current is reduced, at least 400-fold more than transient sodium current is reduced, at least 500-fold more than transient sodium current is reduced, at least 600-fold more than transient sodium current is reduced, at least 700-fold more than transient sodium current is reduced, at least 800-fold more than transient sodium current is reduced, at least 900-fold more than transient sodium current is reduced or at least 1000-fold more than transient sodium current is reduced.
Aspects of the present invention provide, in part, a test sample and a control sample. As used herein, the term “test sample” means a sample comprising a potential persistent Na+ channel blocker. As used herein, the term “potential persistent Na+ channel blocker” means any molecule that is to be tested for its ability to reduce or prevent a persistent Na+ current derived from at least one persistent Na+ channel. A potential persistent Na+ channel blocker can be an inorganic molecule or an organic molecule. As used herein, the term “control sample” means a sample of the same or similar type as the test sample under the same conditions but which does not contain a potential persistent Na+ channel blocker. In addition, a control sample may comprise a defined molecule known not to be a persistent Na+ channel blocker (a negative control molecule) or a defined molecule known to be a persistent Na+ channel blocker (a positive control molecule). One skilled in the art understands that a variety of control samples are useful in the methods of the invention and that a control sample can be a positive control sample or a negative control sample.
Aspects of the present invention provide, in part, a physiological solution. As used herein, the term “physiological solution” means a solutioned solution comprising physiological concentrations of sodium, potassium, magnesium, calcium and chloride. It is also envisioned that any and all physiological solutions that allow an electrical current to be measured from the solution can be used in methods disclosed in the present specification. Optionally, a physiological solution can contain inhibitors for other ion conductances, such as, e.g., a concentration of cadmium that prevents or reduces calcium current. A physiological solution can be varied as appropriate by one skilled in the art and generally depend, in part, on the assay protocol, the cell or the detection method employed. Ion concentration can be varied as appropriate by one skilled in the art and generally depend, in part, on the buffering capacity of a particular buffer being used and the detection means employed.
Aspects of the present invention provide, in part, a Na+-free physiological solution. As used herein, the term “Na+-free physiological solution” means a buffered solution comprising physiological concentrations of a non-permeant sodium substitute, potassium, magnesium, calcium and chloride. It is also envisioned that any and all Na+-free physiological solutions that allow an electrical current to be measured from the solution can be used in methods disclosed in the present specification. A non-permeant sodium substitute substitutes Na+ with an analog cation molecule, such as, e.g., TEA or NMDG+. It is also envisioned that any and all Na+-free physiological solutions that allow an electrical current to be measured from the solution can be used in methods disclosed in the present specification. Optionally, a Na+-free solution can contain inhibitors for other ion conductances, such as, e.g., a concentration of cadmium that prevents or reduces calcium current. A Na+-free physiological solution can be varied as appropriate by one skilled in the art and generally depend, in part, on the assay protocol, the cell or the detection method employed. Ion concentration can be varied as appropriate by one skilled in the art and generally depend, in part, on the buffering capacity of a particular buffer being used and the detection means employed.
Aspects of the present invention provide, in part, a Cl−-free physiological solution. As used herein, the term “Cl−-free physiological solution” means a buffered solution comprising physiological concentrations of a non-permeant chloride substitute, potassium, magnesium, calcium and sodium. It is also envisioned that any and all Cl−-free physiological solutions that allow an electrical current to be measured from the solution can be used in methods disclosed in the present specification. A non-permeant chloride substitute substitutes Cl− with an analog molecule, such as, e.g., gluconate, aspartate, glutamate, cyclamate and methanesulfonate. It is also envisioned that any and all Cl−-free physiological solutions that allow an electrical current to be measured from the solution can be used in methods disclosed in the present specification. Optionally, a Cl−-free solution can contain inhibitors for other ion conductances, such as, e.g., a concentration of cadmium that prevents or reduces calcium current. A Cl−-free physiological solution can be varied as appropriate by one skilled in the art and generally depend, in part, on the assay protocol, the cell or the detection method employed. Ion concentration can be varied as appropriate by one skilled in the art and generally depend, in part, on the buffering capacity of a particular buffer being used and the detection means employed.
A physiological solution, Na+-free physiological solution or Cl−-free physiological solution can be buffered, e.g., 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffers; Phosphate buffers, such as, e.g., potassium phosphate buffers and sodium phosphate buffers; Good buffers, such as, e.g., piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), N,N′-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino) propanesulfonic acid (MOPS), N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO), N-tris(hydroxymethyl) methylglycine (Tricine), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS); saline buffers, such as, e.g., Phosphate-buffered saline (PBS), HEPES-buffered saline, Tris-buffered saline (TBS) and Ringer's. Thus, aspects of this embodiment may include a buffer concentration of, e.g., at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, or at least 100 mM. Non-limiting examples of how to make and use specific buffers are described in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra, (2001).
Aspects of the present invention provide, in part, a voltage-sensitive fluorescent dye. The plasma membrane of a cell typically has a transmembrane potential of approximately −70 mV (negative inside) as a consequence of K+, Na+ and Cl− concentration gradients maintained by active transport processes. Voltage-sensitive fluorescent dyes can directly measure changes in membrane potential resulting from the translocation of these ions. It is envisioned that any voltage-sensitive florescent dye capable of detecting a change in cell membrane potential can be used, including, without limitation, coumarin dyes, such as, e.g., N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine (CC2-DMPE); anionic and hybrid oxonol dyes, such as, e.g., bis-oxonol, oxonol V (bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethine oxonol), oxonol VI (bis-(3-propyl-5-oxoisoxazol-4-yl) pentamethine oxonol), bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3), bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5), RH-155 (NK3041), RH-479 (JPW1131), RH-482 (JPW1132, NK3630), RH-1691, RH-1692, RH-1838 R-1114 (WW781), JPW1177 and JPW1245; hemicyanine dyes, such as, e.g., dibutylamino-naphtalene-butylsulfonato-isoquinolinium (BNBlQ); merocyanine dyes, such as, e.g., merocyanine 540, NK2495 (WW375) and JPW1124; cationic or zwitterionic styryl dyes, such as, e.g., di-4-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-4-ANEPPS), di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), di-12-ANEPPS, di-18:2-ANEPPS, di-2-ANEPEQ (JPW1114), di-12-ANEPEQ, di-8-ANEPPQ, di-12-ANEPPQ, di-1-ANEPIA, D-6923 (JPW3028), N-(4-sulfobutyl)-4-(6-(4-(dibutylamino)phenyl)hexatrienyl) pyridinium (RH-237), N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl) pyridinium dibromide (RH-414), N-(4-sulfobutyl)-4-(4-(4-(dipentylamino)phenyl)butadienyl) pyridinium (RH-421) and RH-437, RH-461, RH-795, JPW1063 and FM1-43; and cationic carbocyanines and rhodamines, such as, e.g., 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)), 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)), 3,3′-dimethylnaphthoxacarbocyanine iodide (JC-9; DiNOC1(3)), 3,3′-dipentyloxacarbocyanine iodide (DiOC5(3), 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)), 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (DilC1(5)), rhodamine, rhodamine 123, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (CBlC2(3), tetramethylrhodamine, ethyl ester, perchlorate (TMRE) and tetramethylrhodamine, methyl ester, perchlorate (TMRM). The class of dye determines factors such as accumulation in cells, response mechanism and toxicity.
Voltage-sensitive fluorescent dyes can also be divided into two general categories of based on whether there is a relatively fast intramolecular redistribution of electrons or a relatively slow transmembrane movement of entire dye (Table 1). Fast-response dyes undergo electric field-driven changes of intramolecular charge distribution in response to a change in the surrounding electric field. This change in electronic structure produce corresponding changes in the spectral properties or intensity of their fluorescence. The optical response of these dyes is sufficiently fast to detect transient (millisecond) potential changes in excitable cells, including single neurons, cardiac cells and intact brains. However, the magnitude of their potential-dependent fluorescence change is often small; fast-response probes typically show a 2-10% fluorescence change per 100 mV. Non-limiting examples of Fast-response dyes include, e.g., di-2-ANEPEQ (JPW1114), di-1-ANEPIA, di-8-ANEPPQ, di-12-ANEPPQ, di-4-ANEPPS, di-8-ANEPPS, di-18:2-ANEPPS, RGA-30, RH-155, RH-795, RH-237, RH-421, RH-414 and WW 781.
Slow-response dyes are lipophilic anions or cations that are exhibit potential-dependent changes in their transmembrane distribution by an electrophoretic mechanism. Fluorescence changes associated with transmembrane redistribution result from sensitivity of the dye to intracellular and extracellular environments. The magnitude of their optical responses is much larger than that of fast-response probes (typically a 1% fluorescence change per mV). Slow-response probes, which include cationic carbocyanines and rhodamines and anionic oxonols, are suitable for detecting changes in average membrane potentials of non-excitable cells caused by respiratory activity, ion-channel permeability, drug binding and other factors. Non-limiting examples of Slow-response dyes include, e.g., DiSBAC4(3), DiBAC4(5), DiBAC4(3), DiOC5(3), DiOC6(3), DiSC3(5), DiOC2(3), DiNOC1(3), DilC1(5), merocyanine 540, Oxonol V, Oxonol VI, rhodamine 123, TMRM, TMRE and CBlC2(3).







TABLE 1







Voltage-sensitive fluorescent dyes










Dye
Response
Absorbance
Emission





di-2-ANEPEQ (JPW1114)
Fast
517
721


di-1-ANEPIA
Fast
—
—


di-8-ANEPPQ
Fast
516 (467)
721 (631)


di-12-ANEPPQ
Fast
519
719


di-1-ANEPMI
Fast
—
—


di-4-ANEPPS
Fast
497 (475)
705 (617)


di-8-ANEPPS
Fast
498
713


di-18:2-ANEPPS
Fast
501
705


RGA-30
Fast
629
659


RH-155
Fast
650
none


RH-795
Fast
530
712


RH-237
Fast
528 (506)
782 (687)


RH-421
Fast
515 (493)
704 (638)


RH-414
Fast
532
716


WW781
Fast
605
639


DiSBAC4(3)
Slow
535
560


DiBAC4(5)
Slow
590
616


DiBAC4(3)
Slow
493
516


DiOC5(3)
Slow
484
500


DiOC6(3)
Slow
484
501


DiSC3(5)
Slow
651
675


DiOC2(3)
Slow
482
497


DiNOC1(3)
Slow
522
535


DiIC1(5)
Slow
638
658


merocyanine 540
Slow
555
578


Oxonol V
Slow
610
639


Oxonol VI
Slow
599
634


rhodamine 123
Slow
507
529


TMRM
Slow
549
573


TMRE
Slow
549
574


CBIC2(3)
Slow
514
529





Spectra values are in methanol with values in parenthesis in a membrane environment. Absorbance and emission spectra of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and 80 nm for emission, but varies considerably from one dye to another. Styryl dyes are generally nonfluorescent in water.






Voltage-sensitive fluorescent dyes have been widely used to monitor membrane potential within neuronal and other cell types, see, e.g., Amiram Grinvald et al., Optical imaging of neuronal activity, 68(4) Physiol. Rev. 1285-1366 (1988); C. R. Lowe and M J. Goldfinch, Solid-phase optoelectronic biosensors, 137 Methods Enzymol. 338-348 (1988); and Haralambos E. Katerinopoulos, The coumarin moiety as chromophore of fluorescent ion indicators in biological systems, 10(30) Curr. Pharm. Des. 3835-3852 (2004). Voltage-sensitive fluorescent dye with high sensitivity and rapidly response to a change in membrane potential and methods for measuring membrane potential using such dyes are well known to those skilled in the art, and are described in, e.g., lain D. Johnson, Fluorescent Probes for Living Cells 30(3) HISTOCHEM. J. 123-140 (1998); and IMAGING NEURONS: A LABORATORY MANUAL (Rafael Yuste, et al., eds., Cold Spring Harbor Laboratory Press, 2000). In addition, the methods disclosed in the present specification can take advantage of the high temporal and spatial resolution utilized by fluorescence resonance energy transfer (FRET) in the measurement of membrane potential by voltage-sensitive dyes as described, see, e.g., Jesus E. Gonzalez & Roger Y. Tsien, Improved Indicators of Cell Membrane Potential That Use Fluorescence Resonance Energy Transfer 4(4) CHEM. BIOL. 269-277 (1997); Roger Y. Tsien & Jesus E. Gonzalez, Voltage Sensing by Fluorescence Resonance Energy Transfer, U.S. Pat. No. 5,661,035 (Aug. 26, 1997); Roger Y. Tsien & Jesus E. Gonzalez, Detection of Transmembrane Potentials by Optical Methods, U.S. Pat. No. 6,107,066 (Aug. 22, 2000).
It is also envisioned that assays involving Fluorescence Resonance Energy Transfer (FRET) can be used to detect a change in cell membrane potential. FRET is a distance dependent interaction between the electronic excited states of two molecules in which excitation is transferred from a donor fluorophore to an acceptor without emission of a photon. The process of energy transfer results in a reduction (quenching) of fluorescence intensity and excited state lifetime of the donor fluorophore and, where the acceptor is a fluorophore, can produce an increase in the emission intensity of the acceptor. Upon induction of a persistent Na+ current, the membrane is depolarized, resulting a separation of the donor/acceptor pair and thus the resonance energy transfer is reduced and can be detected, for example, by increased donor fluorescence emission, decreased acceptor fluorescence emission, or by a shift in the emission maxima from near the acceptor emission maxima to near the donor emission maxima. In the presence of a persistent current blocker, membrane depolarization and thus changes in FRET are reduced or prevented. If desired, the amount of persistent Na+ current reduction or prevention, modulated by a persistent Na+ channel, can be calculated as a function of the difference in the degree of FRET using the appropriate standards.
As a non-limiting example, a FRET pair comprises a voltage-sensitive mobile acceptor DiSBAC2(3) and a fluorescent, membrane-bound donor CC2-DMPE. When the cell interior has a relatively negative potential, the DiSBAC2(3) will bind to the exterior of the cell membrane, resulting in efficient FRET. When the cell interior has a relatively positive potential, however, the DiSBAC2(3) will bind to the interior of the cell membrane, thus separating the FRET pair and disrupting FRET. Other non-limiting examples of fluorophores useful as acceptors for the CC2-DMPE donor are listed in Table 2.







TABLE 2







Donor Fluorophores and Acceptors










Donor
Acceptor







CC2-DMPE
DiSBAC2(3)



CC2-DMPE
DiSBAC4(3)



CC2-DMPE
RH-155 (NK3041)



CC2-DMPE
RH-479 (JPW1131)



CC2-DMPE
RH-482(JPW1132, NK3630)



CC2-DMPE
RH-1691



CC2-DMPE
RH-1692



CC2-DMPE
RH-1838



CC2-DMPE
R-1114(WW781)



CC2-DMPE
JPW1177



CC2-DMPE
JPW1245










Aspects of the present invention provide, in part, a cell. As used herein, the term “cell,” means any cell that natively expresses the molecules necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel a K+ channel or a Na/K ATPase pump, or can be genetically engineered to express the molecules necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel a K+ channel or a Na/K ATPase pump. As a non-limiting example, a cell useful for practicing a method using a Na+ depletion/repletion protocol would be a cell that natively express a K+ channel, a transient Na+ channel and a persistent Na+ channel, or a cell genetically engineered to express a K+ channel, a transient Na+ channel and a persistent Na+ channel. As another non-limiting example, a cell useful for practicing a method using a hyperpolarization protocol would be a cell that natively express a K+ channel and a persistent Na+ channel, or a cell genetically engineered to express a K+ channel and a persistent Na+ channel. As yet another non-limiting example, a cell useful for practicing a method using a Na/K ATPase pump inhibitor protocol would be a cell that natively express a persistent Na+ channel and Na/K ATPase pump, or a cell genetically engineered to express a persistent Na+ channel and Na/K ATPase pump.
A cell can be obtained from a variety of organisms, such as, e.g., murine, rat, porcine, bovine, equine, primate and human cells; from a variety of cell types such as, e.g., neural and non-neural; and can be isolated from or part of a heterogeneous cell population, tissue or organism. It is understood that cells useful in aspects of the invention can include, without limitation, primary cells; cultured cells; established cells; normal cells; transformed cells; tumor cells; infected cells; proliferating and terminally differentiated cells; and stably or transiently transfected cells. It is further understood that cells useful in aspects of the invention can be in any state such as proliferating or quiescent; intact or permeabilized such as through chemical-mediated transfection such as, e.g., calcium phosphate-mediated, diethylaminoethyl (DEAE) dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated, polybrene-mediated, and protein delivery agents; physical-mediated tranfection, such as, e.g., biolistic particle delivery, microinjection and electroporation; and viral-mediated transfection, such as, e.g., retroviral-mediated transfection. It is further understood that cells useful in aspects of the invention may include those which express a Na+ channel under control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both.
Naturally occurring cells having persistent sodium current include, without limitation, neuronal cells, such as, e.g., squid axon, cerebellar Purkinje cells, neocortical pyramidal cells, thalamic neurons, CA1 hipppocampal pyramidal cells, striatal neurons and mammalian CNS axons. Other naturally occurring cells having persistent sodium current can be identified by those skilled in the art using methods disclosed herein below and other well known methods. Genetically engineered cells expressing a persistent Na+ current can include, without limitation, isolated mammalian primary cells; established mammalian cell lines, such as, e.g., COS, CHO, HeLa, NIH3T3, HEK 293-T and PC12; amphibian cells, such as, e.g., Xenopus embryos and oocytes; insect cells such as, e,g,, D. melanogaster; yeast cells such as, e.g., S. cerevisiae, S. pombe, or Pichia pastoris and prokaryotic cells, such as, e.g., E. coli. 
Cells can be genetically engineered to express a polynucleotide molecule encoding a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel a K+ channel or a Na/K ATPase pump. The sequences of polynucleotide molecules encoding a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel a K+ channel or a Na/K ATPase pump are well-known and publicly available to one skilled in the art. For example, both polynucleotide and protein sequences of all currently described persistent Na+ channels, transient Na+ channels, K+ channels and Na/K ATPase pumps are publicly available from the GenBank database (National Institutes of Health, National Library of Medicine. In addition, polynucleotide and protein sequences are described, see, e.g, Alan L. Goldin, Diversity of Mammalian Voltage-gated Sodium Channels, 868 ANN. N.Y. ACAD. SCI. 38-50 (1999), William A. Catterall, From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-gated Sodium Channels, 26(1) NEURON 13-25 (2000); John N. Wood & Mark D. Baker, Voltage-gated Sodium Channels, 1(1) CURR. OPIN. PHARMACOL. 17-21 (2001); and Frank H. Yu & William A. Catterall, Overview of the Voltage-Gated Sodium Channel Family, 4(3) GENOME BIOL. 207 (2003).
Voltage-gated Na+ channels are members of a large mammalian gene family encoding at least nine alpha- (Nav 1.1-Nav 1.9) and four beta-subunits. While all members of this family conduct Na+ ions through the cell membrane, they differ in tissue localization, regulation and, at least in part, in kinetics of activation and inactivation, see, e.g., Catterall, supra, (2000); and Sanja D. Novakovic et al., Regulation of Na+ Channel Distribution in the Nervous System, 24(8) TRENDS NEUROSCI. 473-478 (2001). Four sodium channels, Nav 1.3, Nav 1.5, Nav 1.6 and Nav 1.9, have historically been known to generate a persistent current. Recent evidence, however, suggests that all voltage-gated sodium channels are capable of producing a persistent current, see, e.g., Abraha Taddese & Bruce P. Bean, Subthreshold Sodium Current from Rapidly Inactivating Sodium Channels Drives Spontaneous Firing of Tubermammillary Neurons, 33(4) NEURON 587-600 (2002); Michael Tri H. Do & Bruce P. Bean, Subthreshold Sodium Currents and Pacemaking of Subthalamic Neurons: Modulation by Slow Inactivation, 39(1) NEURON 109-120 (2003). As of Nov. 21, 2005, accession numbers for representative human voltage-gated Na+ channel family members include gi29893559, gi10337597, gi25014054, gi40255316, gi37622907, gi7657544, gi4506813, gi56748895 and gi7657542, which are hereby incorporated by reference in their entirety.
Voltage-gated K+ channels are members of a large mammalian gene family encoding at least 5 six transmembrane subunits: Kv 1.x, Kv 2.x, Kv 3.x, Kv 4.x and KvCNQ, see, e.g., Gary Yellen, The voltage-gated potassium channels and their relatives, 419(6902) Nature 35-42 (2002). These ion channels help maintain and regulate the K+-based component of the membrane potential and are thus central to many critical physiological processes. Each subunit family is composed of several genes. Thus, the Kv 1.x family in mammals is comprised of five distinct genes: Kv 1.1, Kv 1.2, Kv 1.3, Kv 1.4 and Kv 1.5. The Kv 2.x family in mammals is comprised of two distinct genes: Kv 2.1 and Kv 2.2. The Kv 3.x family in mammals is comprised of four distinct genes: Kv 3.1, Kv 3.2a, Kv 3.2b, Kv 3.2c, Kv 3.3, Kv 3.4a and Kv 3.4b. The Kv 4.x family in mammals is comprised of three distinct genes: Kv 4.1, Kv 4.2, Kv 4.3-1 and Kv 4.3-2. As of Nov. 21, 2005, accession numbers for representative human voltage-gated K+ channel family members include Kv 1.x channels gi4557685, gi4826782, gi25952082, gi4504817 and gi25952087; Kv 2.x channels gi4826784 and gi27436974; Kv 3.x channels gi76825377, gi21217561, gi21217563, gi24497458, gi24497460, gi24497462 and gi24497464; and Kv 4.x channels gi27436981, gi9789987 and gi27436984, gi27436986, which are hereby incorporated by reference in their entirety.
The Na/K ATPase pump family is a member of the P-type ATPase superfamily. Two subunits α and β comprise the Na/K pump. In mammals, four a isoforms have been identified (α1, α2, α3, α4). A housekeeping function is assigned to α1. This isoform is expressed throughout the body. The α2 isoform is expressed mainly in brain, heart and skeletal muscle and appears to be involved in regulation cell Ca2+. The α4 isoform is believed to help maintain sperm motility. The Na/K ATPase pump family in mammals is comprises Na/K α1a, Na/K α1b, Na/K α2, Na/K α3 and Na/K α4. As of Nov. 21, 2005, accession numbers for representative human Na/K ATPase pump family members include gi21361181, gi48762682, gi1703467, gi29839750 and gi37577153, which are hereby incorporated by reference in their entirety.
Another aspect of the present invention provides, in part, an expression construct that allow for expression of a polynucleotide molecule encoding a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel, a K+ channel or a Na/K ATPase pump. These expression constructs comprise an open reading frame encoding a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel, a K+ channel or a Na/K ATPase pump, operably-linked to control sequences from an expression vector useful for expressing a the necessary molecule in a cell. The term “operably linked” as used herein, refers to any of a variety of cloning methods that can ligate a polynucleotide molecule disclosed in the present specification into an expression vector such that a polypeptide encoded by the composition is expressed when introduced into a cell. Well-established molecular biology techniques that may be necessary to make an expression construct disclosed in the present specification including, but not limited to, procedures involving polymerase chain reaction (PCR) amplification restriction enzyme reactions, agarose gel electrophoresis, nucleic acid ligation, bacterial transformation, nucleic acid purification, nucleic acid sequencing are routine procedures well within the scope of one skilled in the art and from the teaching herein. Non-limiting examples of specific protocols necessary to make an expression construct are described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.
A wide variety of expression vectors can be employed for expressing an open reading frame encoding a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel, a K+ channel or a Na/K ATPase pump, and include without limitation, viral expression vectors, prokaryotic expression vectors and eukaryotic expression vectors including yeast, insect and mammalian expression vectors. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.
It is envisioned that any of a variety of expression systems may be useful for expressing a construct disclosed in the present specification. An expression system encompasses both cell-based systems and cell-free expression systems. Cell-based systems include, without limited, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Cell-free systems include, without limitation, wheat germ extracts, rabbit reticulocyte extracts and E. coli extracts. Expression using an expression system can include any of a variety of characteristics including, without limitation, inducible expression, non-inducible expression, constitutive expression, viral-mediated expression, stably-integrated expression, and transient expression. Expression systems that include well-characterized vectors, reagents, conditions and cells are well-established and are readily available from commercial vendors that include, without limitation, Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science, Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limiting examples on the selection and use of appropriate heterologous expression systems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Hames eds., Oxford University Press, 1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai & Harish Padh, Expression Systems for Production of Heterologous Proteins, 80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.
An expression construct comprising a polynucleotide molecule encoding a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel, a K+ channel or a Na/K ATPase pump, can be operationally-linked to a variety of regulatory elements that can positively or negatively modulate, either directly or indirectly, the expression of a polynucleotide molecule, such as, e.g., constitutive, tissue-specific, inducible or synthetic promoters and enhancers. Using such systems, one skilled in the art can express the desired levels of a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel, a K+ channel or a Na/K ATPase pump, using routine laboratory methods as described, see, e.g., Molecular Cloning A Laboratory Manual (Joseph Sambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press, 3rd ed. 2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds., John Wiley & Sons, 2004). Non-limiting examples of constitutive regulatory elements include, e.g., the cytomegalovirus (CMV), herpes simplex virus thymidine kinase (HSV TK), simian virus 40 (SV40) early, 5′ long terminal repeat (LTR), elongation factor-1α (EF-1α) and polybiquitin (UbC) regulatory elements. Non-limiting examples of inducible regulatory elements useful in aspects of the present invention include, e.g., chemical-inducible regulatory elements such as, without limitation, alcohol-regulated, tetracycline-regulated, steroid-regulated, metal-regulated and pathogenesis-related; and physical-inducible regulatory elements such as, without limitation, temperature-regulated and light-regulated. Such inducible regulatory elements can be prepared and used by standard methods and are commercially available, including, without limitation, tetracycline-inducible and tetracycline-repressible elements such as, e.g., Tet-OnTM and Tet-OffTM (BD Biosciences-Clontech, Palo Alto, Calif.) and the T-RExTM (Tetracycline-Regulated Expression) and Flp-InTM T-RExTM systems (Invitrogen, Inc., Carlsbad, Calif.); ecdysone-inducible regulatory elements such as, e.g., the Complete Control® Inducible Mammalian Expression System (Stratagene, Inc., La Jolla, Calif.); isopropyl β-D-galactopyranoside (IPTG)-inducible regulatory elements such as, e.g., the LacSwitch® II Inducible Mammalian Expression System (Stratagene, Inc., La Jolla, Calif.); and steroid-inducible regulatory elements such as, e.g., the chimeric progesterone receptor inducible system, GeneSwitchTM (Invitrogen, Inc., Carlsbad, Calif.). The skilled person understands that these and a variety of other constitutive and inducible regulatory systems are commercially available or well known in the art and can be useful in the invention for controlling expression of a polynucleotide which encodes a molecule necessary to practice a method disclosed in the present specification, such as, e.g., a persistent Na+ channel, a transient Na+ channel, a K+ channel or a Na/K ATPase pump.
Aspects of the present invention provide, in part, cells comprising a certain gK/gNa ratio, or relative gK/gNa conductance. The relative gK/gNa conductance of genetically engineered cells can be measured simply by increasing the extracelluar K+ concentration and monitoring the change in membrane potential. Contributions of other ions, such as, e.g., Cl−, to the overall membrane potential can be controlled by substituting non-permeant analogs or pharmacological blockers to prevent their contribution to the equilibrium potential. The relative gK/gNa conductance can be calculated using the modified form of the Chord Conductance equation below:

ΔEm=λ(EK2−EK1)/(λ+1)

Where: λ=GK/GNa; EK2=the equilibrium potential for K+ following a 10-fold increase in extracellular K+; EK1=the equilibrium potential for K prior to increasing K+ 10-fold.
For any ion the equilibrium potential is defined as

Ei=(RT/ZF)log[Iout/Iin]

Where: at physiological temperature, with monovalent ions, RT/ZF=60 mV; and Iout and Iin are the concentrations of the relevant ion in the extracellular and intracellular compartments respectively.
As a non-limiting example, to determine the relative gK/gNa conductance for a particular cell line, a 10-fold increase in extracellular K+ concentration is added to the physiological solution. This added K+ induces a depolarization by shifting the equilibrium potential by approximately 60 mV in the positive direction. The values obtained from the experiment can then be used in the modified form of the Chord Conductance equation above to calculate the relative gK/gNa conductance. For a Na/K ATPase pump inhibitor protocol, a relative gK/gNa conductance ≧20-fold is indicative of a depolarization near the theoretical equilibrium potential 60 mV, and thus suitable for this protocol.
Aspects of the present invention provide, in part, detecting fluorescence emitted by the voltage-sensitive dye. The fluorescence emitted from a sample is typically determined using a fluorimeter. In fluorescence detection relying on a single fluorophore, an excitation radiation from an excitation source passes through excitation optics and excites the voltage-sensitive dye. In response, voltage-sensitive dye emits radiation which has an emission wavelength that is different from the excitation wavelength, which is collected by collection optics. In fluorescence detection relying on FRET, an excitation radiation from an excitation source having a first wavelength passes through excitation optics. The excitation optics cause the excitation radiation to excite the voltage-sensitive dye. In response, voltage-sensitive dye emits radiation which has an emission wavelength that is different from the excitation wavelength, which is collected by collection optics. If desired, the device includes a temperature controller to maintain the cell at a specific temperature while being scanned. If desired, a multi axis translation stage moves a microtiter plate containing a plurality of samples in order to position different wells to be exposed. It is understood that the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by the appropriate digital computer.
It is further understood that the methods of the invention can be automated and can be configured in a high throughput or ultra high-throughput format using, without limitation, 96 well, 384-well or 1536 well plates. Instrumentation useful for measuring membrane potential for high-throughput screening procedures include, without limitation, Fluorometric Imaging Plate Reader (FLIPR®; Molecular Devices, Sunnyvale, Calif.) and Voltage/Ion Probe Reader (VIPR; Aurora Biosciences, San Diego, Calif.), see, e.g., Roger Y. Tsien & Jesus E. Gonzalez, Detection of Transmembrane Potentials by Optical Methods, U.S. Pat. No. 6,342,379 (Jan. 29, 2002); Jesus E. Gonzalez & Michael P. Maher, Cellular Fluorescent Indicators and Voltage/Ion Probe Reader (VIPR) Tools for Ion Channel and Receptor Drug Discovery, 8(5-6) RECEPTORS CHANNELS 283-295, (2002); and Michael P. Maher & Jesus E. Gonzalez, High Throughput Method and System for Screening Candidate Compounds for Activity Against Target Ion Channels, U.S. Pat. No. 6,686,193 (Feb. 3, 2004). As a non-limiting example, fluorescence emission can be detected using the FLIPR® instrumentation system, which is designed for 96-well plate assays. FLIPR® utilizes a water-cooled 488 nm argon ion laser (5 watt) or a xenon arc lamp and a semiconfocal optical system with a charge coupled device (CCD) camera to illuminate and image the entire plate. The FPM-2 96-well plate reader (Folley Consulting and Research; Round Lake, Ill.) also can be useful in detecting fluorescence emission in the methods of the invention. One skilled in the art understands that these and other automated systems with the appropriate spectroscopic compatibility can be useful in high-throughput screening methods disclosed in the present specification.
Aspects of the present invention provide, in part, determining a relative emitted fluorescence. A relative emitted fluorescence is determined by comparing the fluorescence emitted from a test sample to the corresponding control sample for that test sample. A decrease in emitted fluorescence from a test sample relative to a control sample is indicative of a reduction or prevention of a persistent Na+ current, i.e., the presence of a persistent Na+ channel blocker in the test sample. As a non-limiting example, a decrease in emitted fluorescence from a test sample relative to a control sample using a Na+ depletion/repletion protocol is indicative of a reduction or prevention of a persistent Na+ current, i.e., the presence of a persistent Na+ channel blocker in the test sample. As another non-limiting example, a decrease in emitted fluorescence from a test sample relative to a control sample using a hyperpolarization protocol is indicative of a reduction or prevention of a persistent Na+ current, i.e., the presence of a persistent Na+ channel blocker in the test sample. As yet another non-limiting example, a decrease in emitted fluorescence from a test sample relative to a control sample using a Na/K ATPase pump inhibitor protocol is indicative of a reduction or prevention of a persistent Na+ current, i.e., the presence of a persistent Na+ channel blocker in the test sample.
In an embodiment, a decrease in emitted fluorescence from a test sample relative to a control sample is indicative of a reduction or prevention of a persistent Na+ current, i.e., the presence of a persistent Na+ channel blocker. In aspects of this embodiment, a decreased relative emitted fluorescence from a test sample can be, e.g., at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least ten-fold, at least twenty-fold or more as compared to the relative emitted fluorescence from a control sample. In other aspects of this embodiment, a decreased relative emitted fluorescence from a test sample can be, e.g., at most two-fold, at most three-fold, at most four-fold, at most five-fold, at most ten-fold, at most twenty-fold as compared to the relative emitted fluorescence from a control sample.
In another embodiment, an increase is emitted fluorescence from a control sample relative to a test sample is indicative of a reduction or prevention of a persistent Na+ current, i.e., the presence of a persistent Na+ channel blocker. In aspects of this embodiment, an increased relative emitted fluorescence from a control sample can be, e.g., at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least ten-fold, at least twenty-fold or more as compared to the relative emitted fluorescence from a test sample. In other aspects of this embodiment, an increased relative emitted fluorescence from a control sample can be, e.g., at most two-fold, at most three-fold, at most four-fold, at most five-fold, at most ten-fold, at most twenty-fold as compared to the relative emitted fluorescence from a test sample.
Aspects of the present invention provide a method for identifying a selective blocker of a persistent Na+ channel using a Na+ depletion/repletion protocol (FIG. 2). This protocol relies on the essential requirement of Na+ to generate a persistent current. Generally, cells having a K+ channel, a transient Na+ channel and a persistent Na+ channel are incubated in a Na+-free physiological solution. A small depolarization of the membrane of the cells is induced to activate both transient and persistent Na+ channels. However, no additional depolarization will be observed in the absence of extracellular Na+. Thus, within a few milliseconds following depolarization the transient Na+ channels will close, but the channels capable of generating a persistent Na+ current will remain open. The addition of Na+ to the Na+-free physiological solution will cause these opened persistent Na+ channels to generate a persistent current causing the membrane to depolarize. The presence of a persistent Na+ channel blocker will either eliminate or reduce the magnitude of this depolarization event (FIG. 3). Therefore, screens based on the Na+ depletion/repletion protocol can identify a potential persistent Na+ channel blocker by absent or reduction of membrane depolarization. Conversely, molecules that lack this blocking capability, whether a potential test molecule or a control sample, will not affect the magnitude of this depolarization event. The Na+ depletion/repletion protocol therefore allows the discovery of molecules that block the persistent Na+ current and as such is a screen for persistent Na+ channels blockers.
In one embodiment, a Na+ depletion/repletion protocol test sample comprises a cell comprising a K+ channel, a transient Na+ channel and a persistent Na+ channel.
Aspects of the present invention provide, in part, depolarizing a membrane of the cell. A cell membrane may be depolarized by adding K+ to the medium to shift the K+ equilibrium potential in the positive direction. As an non-limiting example, in a cell in which the K+ conductance dominates at the resting potential and the intracellular and extracellular K+ concentrations are 120 mM and 4.5 mM respectively the equilibrium potential for potassium would be approximately −84 mV. Addition of K+ to bring the extracellular K+ to 13 mM would result in a equilibrium potential for potassium of approximately −57 mV. Depending on the relative contribution of the other ionic conductances of the cell membrane this increase in K+ could result in depolarization of up to 27 mV. One skilled in the art will recognize that there are many other methods for depolarizing a cell membrane. As additional non-limiting examples it would be possible to depolarize the cell membrane by adding K+ channel blockers such as the ions, e.g., Cs+, Ba2+, TEA+, or small organic molecules, e.g., 4-aminopyridine, quinidine or phencyclidine, or peptide toxins e.g. charybdotoxin, margatoxin, iberiotoxin, noxiustoxin, kaliotoxin to the extracellular medium. One skilled in the art would recognize that inhibition of the electrogenic Na+/K+ pump with cardiac glycosides such as ouabain, or and dihydro-ouabain; isothiouronium or derivative thereof, such as, e.g., 1-bromo-2,4,6-tris (methylisothiouronium) benzene (Br-TlTU) and 1,3-dibromo-2,4,6-tris (methylisothiouronium) benzene (Br2-TlTU); digitoxigenin or derivative thereof, such as, e.g., digitalis, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, 22-hydroxy-digitoxigenin and 14β,17β-cycloketoester-3β-OH androstane (lNClCH-D7); coumestan or derivative thereof, such as, e.g., 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36); vanadate or derivative thereof; cardenolide or derivative thereof; and natural cardiac glycosides would depolarize the cell membrane. Additionally, one skilled in the art would recognize that the use of electric field stimulation (EFS) to deliver electrical stimuli to the cell would result in a depolarization of the cell membrane. Each of the above non-limiting examples could be used in combination with each other or other methods to deliver depolarizing stimuli to the cell membrane.
In one embodiment, depolarizing a membrane of the cell can be with the addition of K+. It is envisioned that any K+ concentration can be useful with the proviso that this K+ addition induces a membrane depolarization of at least 5 mV and such addition does not prevent the additional depolarization due to Na+ repletion. For example, a K+-induced depolarization of range of about 5 to about 50 mV. In aspects of this embodiment, the K+-induced depolarization can be, e.g., about 5 mV, about 10 mV, about 20 mV, about 30 mV, about 40 mV or about 50 mV.
Aspects of the present invention provide, in part, generating a current through the persistent Na+ channel by adding a Na+ containing solution into a well containing cells depolarized in the absence Na+. The magnitude of the depolarization will depend on the concentration of Na+ added and the relative conductance of the Na+ channels generating the persistent current. As a non-limiting example addition of Na+ to the extracellular solution that results in a final Na+ concentration of 70-100 mM will result in a robust depolarization of the cell membrane in the presence of persistent sodium channels. In addition, one skilled in the art will recognize that to obtain a relialble measurement of persistent Na+ current a wide range timings for the applications of the Na+ solution would be possible as long as the transient sodium channels were allowed to inactivate.
Aspects of the present invention provide a method for identifying a selective blocker of a persistent Na+ channel using a hyperpolarization protocol (FIG. 4). In this protocol, the proportion of K+ and persistent Na+ channels present in cells is such that their conductances are essentially equal. Assuming all other ion conductances are minimal the resting membrane potential will lie approximately halfway between the equilibrium potential for Na+ and the equilibrium potential of K+. Under these conditions adding a K channel blocker will depolarize the cells toward the equilibrium potential of Na+ (ENa>50 mV). On the other hand, adding a persistent Na+ channel blocker will hyperpolarize the cells driving the membrane potential towards the equilibrium potential for K+ (EK<−85 mV). These predictions can be understood via the chord conductance equation when a cell is solely permeable to Na+ and K+ (the media is Cl−-free and appropriate inhibitors are present as in the Na+ depletion/repletion protocol. Thus, the presence of a persistent Na+ channel blocker generates a hyperpolarization event (FIG. 5). The more potent the persistent Na+ channel blocker the greater the hyperpolarization with complete block bringing the membrane potential to EK. Therefore, screens based on the hyperpolarization protocol can identify a potential persistent Na+ channel blocker by the induction of membrane hyperpolarization. Conversely, molecules that lack this blocking capability, whether a potential test molecule or a control sample, will not induce this hyperpolarization event. The hyperpolarization protocol therefore allows the discovery of molecules that block the persistent Na+ current and as such is a screen for persistent Na+ channels blockers.
In an embodiment, a hyperpolarization protocol test sample comprises a cell comprising a K+ channel and a persistent Na+ channel wherein a resting membrane potential of the cell is approximately halfway between the equilibrium potential of K+ and the equilibrium potential of Na+. In aspects of this embodiment, the resting membrane potential can comprise an approximate range of, e.g., −50 mV to 15 mV, −45 mV to 10 mV, −40 mV to 5 mV, −35 mV to 0 mV, −30 mV to −5 mV, −25 mV to −10 mV or −20 mV to −15 mV. In other aspects of this embodiment, the resting membrane potential can comprise an approximate range of, e.g., −50 mV to −20 mV, −40 mV to −10 mV, −30 mV to 0 mV, −20 mV to 10 mV or −10 mV to 20 mV.
In another embodiment, a hyperpolarization protocol test sample comprises a cell comprising a K+ channel and a persistent Na+ channel wherein a resting membrane potential of the cell is approximately halfway between the equilibrium potential of K+ and the equilibrium potential of Na+ can detect a hyperpolarization of a membrane. In aspects of this embodiment, membrane potential can hyperpolarize by at least 20 mV, at least 30 mV, at least 40 mV, at least 50 mV or at least 60 mV. In other aspects of this embodiment, membrane potential can hyperpolarize by at most 20 mV, at most 30 mV, at most 40 mV, at most 50 mV or at most 60 mV.
Aspects of the present invention provide a method for identifying a selective blocker of a persistent Na+ channel using a Na/K ATPase pump inhibitor protocol, see FIG. 6. This assay relies on the fact that inhibition of the Na/K ATPase will allow net cellular Na+ entry and K loss. In this protocol cells containing persistent Na+ channels, K+ channels, and Na/K ATPase, assayed in a Cl−-free medium physiological solution, are treated with a pump inhibitor. This inhibition will lead to an initial small membrane depolarization due to blockage of the Na/K ATPase pump and a subsequent large secondary depolarization. This secondary depolarization is the key to the assay and relies on the fact that the equilibrium potential for K+ will become more positive as the cells lose K+. The rationale is as follows. For this assay to work GK must be >>GNapersistent. Following addition of a Na/K ATPase pump inhibitor, the cells will gain Na+ via persistent Na+ channels that are open at near resting membrane potential. In the absence of a Cl− conductance the Na+ gained by the cells will be electrically compensated for by an equimolar loss of K+. Since GK>>GNa, the membrane potential will be dominated by K and therefore a decrease in cell K+ will result in a positive change in the potassium equilibrium potential. As a result, a depolarization of the membrane will occur because of millimolar K+ loss. It should be understood that although the cell gains Na+ this gain is of little effect on the membrane potential since GK>>GNa. Instead it is the compensatory movement of K+ ions drives the membrane potential in this assay. Thus, the extent of the depolarization will depend on the amount K+ lost by the cell following the addition of a Na/K ATPase pump inhibitor. The presence of a persistent Na+ channel blocker will either eliminate or reduce the magnitude of this secondary depolarization event (FIG. 7). Therefore, screens based on the Na/K ATPase pump inhibitor protocol can identify a potential persistent Na+ channel blocker by absent or reduction of the secondary depolarization of the membrane. Conversely, molecules that lack this blocking capability, whether a potential test molecule or a control sample, will not affect the magnitude of this depolarization event. The Na/K ATPase pump inhibitor protocol therefore allows the discovery of molecules that block the persistent Na+ current and as such is a screen for persistent Na+ channels blockers.
In an embodiment, a Na/K ATPase pump inhibitor protocol test sample comprises a cell comprising a K+ channel and a persistent Na+ channel wherein a K+ conductance of the K+ channel is greater than the Na+ conductance from the persistent Na+ channel. In aspect of this embodiment, the K+ conductance of the K+ channel is greater than the Na+ conductance by, e.g., at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold In other aspect of this embodiment, the K+ conductance of the K+ channel is greater than the Na+ conductance by, e.g., at most 10-fold higher, at most 20-fold higher, at most 30-fold higher, at most 40-fold higher, at most 50-fold higher or at most 60-fold higher.
Aspects of the present invention provide, in part, a Na/K ATPase pump inhibitor. It is envisioned that any molecule capable of inhibiting the activity of a Na/K ATPase pump can be useful. Non-limiting examples of a Na/K ATPase pump inhibitor include oubain or derivative thereof, such as, e.g., ouabain and dihydro-ouabain; isothiouronium or derivative thereof, such as, e.g., 1-bromo-2,4,6-tris (methylisothiouronium) benzene (Br-TlTU) and 1,3-dibromo-2,4,6-tris (methylisothiouronium) benzene (Br2-TlTU); digitoxigenin or derivative thereof, such as, e.g., digitalis, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, 22-hydroxy-digitoxigenin and 14β, 17β-cycloketoester-3β-OH androstane (lNClCH-D7); coumestan or derivative thereof, such as, e.g., 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36); vanadate or derivative thereof; cardenolide or derivative thereof; and natural cardiac glycosides. The magnitude of the depolarization will depend on the concentration of inhibitor added and the absolute conductance of the Na+ channels generating the persistent current.
In another embodiment, depolarizing a membrane of the cell can be with a Na/K ATPase pump inhibitor. In an embodiment, a Na/K ATPase pump inhibitor depolarizes the cell membrane by inhibiting Na/K ATPase pump activity. In aspects of this embodiment, a Na/K ATPase pump inhibitor used can be, e.g., oubain or derivative thereof, an isothiouronium or derivative thereof, digitoxigenin or derivative thereof, a coumestan or derivative thereof, vanadate or derivative thereof, a cardenolide or derivative thereof or a natural cardiac glycoside.
Aspects of the present invention provide a method for identifying a selective blocker of a persistent Na+ channel using transient Na+ current protocol, see FIG. 8. Protocols such as, e.g., a Na+ depletion/repletion protocol, a hyperpolarization protocol and a Na/K ATPase pump inhibitor protocol, can allow for the identification of a molecule that reduces or prevents a persistent Na+ current. However, these protocols do not address whether the persistent Na+ channel blockers found selectively block persistent Na+ channels, or also block Na+ channels generating the transient current. Thus another part of the screen in accordance with aspects of the present invention addresses how molecules that selectively block persistent Na+ current but not transient Na+ current can be distinguished, i.e., identification of a selective persistent Na+ channel blocker (FIG. 9). In general, a persistent Na+ channel blocker, as determined from a persistent Na+ channel assay, such as, e.g., a Na+ depletion/repletion protocol, a hyperpolarization protocol and a Na/K ATPase pump inhibitor protocol, is retested for its ability to block a transient current. A persistent Na+ channel blocker that is selective for a persistent Na+ channel will not greatly affect transient Na+ current. On the other hand, a persistent Na+ channel blocker that reduces or prevents transient Na+ current as well would not be considered a selective persistent Na+ channel blocker.
It is envisioned that any and all protocols useful for determining a transient Na+ current can be used, including, without limitation, field stimulation. In a field stimulation protocol, electrodes are placed in the well and generate a stimulating current through the cell sufficient to generate an action potential before and after the addition of the persistent Na+ channel blocker. The use of EFS to activate ion channels is a standard procedure well known to one skilled in the art, see, e.g., J. Malmivuo and R. Plonsey, Bioelectromagnetism: Principles and Applications of Bioelectric and Biomagnetic Fields, (Oxford University Press, New York. 1-472 pp. 1995); and J. P. Reilly, Electrical Stimulation and Electropathology (Cambridge University Press, Cambridge. 1-522 pp, 1992). Furthermore, methods to implement these protocols in HTS format have been described, see, e.g., Michael P. Maher & Jesus E. Gonzalez, Multi-well Plate and Electrode Assemblies for Ion Channel Assays, U.S. Pat. No. 6,969,449 (Nov. 29, 2005); and Paul Burnett et al., Fluorescence Imaging of Electrically Stimulated Cells, 8(6) J. Biomol. Screen. 660-667 (2003).
In an embodiment, a field stimulation protocol can depolarize a membrane of the cell.
Aspects of the present invention provide, in part, comparing the emitted fluorescence. Comparisons of emitted fluorescence is achieved by comparing the emitted fluorescence from a persistent Na+ current assay relative to an emitted fluorescence from a transient Na+ current assay for the same potential persistent Na+ channel blocker. With respect to the Na+ depletion/repletion, hyperpolarization and Na/K ATPase pump inhibitor protocols, a decrease is emitted fluorescence from a persistent Na+ current assay relative to a transient Na+ current assay is indicative of a selective reduction or prevention of a persistent Na+ current relative to a transient Na+ current, i.e., the presence of a selective persistent Na+ channel blocker in the test sample.
Although there has been hereinabove described a method and screen for identifying a Na+ channel blocker, in accordance with the present invention, for the purposes of illustrating the manner in which the invention may be used to advantage, it will be appreciated that the invention is not limited thereto. Accordingly, any and all modification, variations or equivalent arrangements which may occur to those skilled in the art should be considered to be within the scope of the invention as defined in the appended claims.
EXAMPLES
Example 1
Screening Assay for Identifying Persistent Sodium Current Blockers Using FRET Technology
To establish an assay plate, HEK-293 cells grown in Minimum Essential Medium (Invitrogen, Inc., Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen, Inc., Carlsbad, Calif.), 1% Pennicillin-Streptomycin (Invitrogen, Inc., Carlsbad, Calif.) were either transiently or stably transfected with a polynucleotide molecule expressing a Nav 1.3 sodium channel capable of mediating persistent sodium current. Stably transfected cells were grown in the presence of 500 mg/mL G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μM TTX (Calbiochem, Inc., San Diego, Calif.) to maintain selective pressure. Cells were grown in vented cap flasks, in 90% humidity and 10% CO2, to about 80% confluence, harvested by trypsinization and cell density was determined. Approximately 16 to 24 hours before the assay, each well of a clear-bottom, black-wall 96-well plate (Becton-Dickinson, San Diego, Calif.) coated with Matrigel (Becton-Dickinson, San Diego, Calif.) was seeded with approximately 75,000 HEK-Nav 1.3 cells in 150 μL of supplemented MEM. Cells were sometimes incubated in 96-well plates at somewhat lower densities (20,000 per well), and incubated for up to 40-48 hours.
To examine the ability of test molecules to alter persistent sodium current, the medium was aspirated, HEK cells were washed 3 times with 150 uL of TEA-MeSO3 solution using CellWash (Thermo LabSystems, Franklin, Mass.) and 150 uL of a Na+-free media and physiologic concentrations of K+ (4.5 mM) was added. Extracellular Cl− was replaced with MeSO3 during preincubation and throughout the assay. This eliminates a complicating Cl− current during the assay and results in an amplified and more stable voltage-change induced by the persistent Na+ current. The HEK cells were preincubated for 30-60 minutes with the ion-sensitive FRET dye CC2-DMPE (final concentration 10 μM). CC2-DMPE is a stationary coumarin-tagged phospholipid resonance energy donor that has an optimal excitation wavelength at approximately 405 nm wavelength light and an optimal emission wavelength at approximately at 460 nm. While the HEK cells were being stained with coumarin, a 10 μM DiSBAC2(3) solution in TEA-MeSO3 solution was prepared. DiSBAC2(3) is a mobile resonance energy acceptor that partition across the membrane as a function of the electric field. The optimal excitation spectra for these dyes overlap the emission spectra of the coumarin donor and, thus, they act as FRET acceptors. DiSBAC2(3) has an emission spectrum in the range of 570 nm. In addition to DiSBAC2(3), this solution contained any test molecule being tested or a DMSO control, at 4 times the desired final concentration (e.g., 20 μM for 5 μM final), 1.0 mM ESS-AY17 to reduce background fluorescence, and 400 μM CdCl2, which stabilizes the membrane potential of the cells at negative resting potential, resulting in the maximum number of Na+ channels being available for activation. After 30-60 minutes of CC2-DMPE staining, the cells were washed 3 times with 150 μL of TEA-MeSO3 solution. Upon removing the solution, the cells were loaded with 80 μL of the DiSBAC2(3) solution and incubated for 20-30 minutes as before. Typically, wells in one column on each plate were free of test drug(s) and served as positive and negative controls.
The assay plates were then transferred to a voltage/ion probe reader (VIPR) (Aurora Biosciences, San Diego, Calif.) and the VIPR was adjusted so that the fluorescent emission ratio from the donor ands acceptor FRET dyes equaled 1.0. To elicit persistent sodium current, a double addition protocol was performed by first adding 240 μL of NaMeSO3 solution to adjust the concentration of sodium and potassium ions in the well to 110 mM and 10 mM, respectively, and measuring the resulting sodium-dependent depolarization and second by adding K+ to a final concentration of 80 mM, and measuring potassium-dependent depolarization. 240 μL of TEA-MeSO3 solution or 1 μM TTX was used as a positive control. Test compounds that block the Na+-dependent signal, but not the K+-dependent signal were selected for further analysis. The Na+-dependent depolarization resulting from the persistent Na+ was measured as shown in FIG. 10. The labeled boxes indicate the application of Na+ or K+. Circles indicate the control response with 0.1% DMSO added, triangles show the effects of the Na+ channel inhibitor tetracaine (10 μM), and the diamonds show the response during the application of a non-specific channel blocker.
In this high-throughput assay, non-specific blockers that inhibit membrane depolarization induced by any effector must be distinguished from selective persistent Na+ current blockers, which block only persistent Na+-dependent depolarizations. Therefore, a counter-screen to determine the ability of compounds to alter K+ dependent depolarization was performed. As shown in FIG. 10, following preincubation with vehicle alone (DMSO) both Na+ and K+ additions produced a robust depolarization as indicated by the increase in Rf/Ri. Tetracaine, a Na+ channel blocker, inhibited the Na+-dependent, but not the K+-dependent change in Rf/Ri. In contrast, a non-specific inhibitor of Na+ and K+-dependent depolarization blocked the change in Rf/Ri following either addition. This data demonstrates that selective blockers of the persistent sodium current can be identified using the described method.
To eliminate compounds that non-specifically inhibited the Na+-dependent depolarization, data obtained using the above procedure were analyzed with respect to a counter-screen that used K+-dependent depolarization as a readout. To select hits from the primary screen, the data were plotted as histograms. Inhibition of the Na+-dependent depolarization was plotted against inhibition of the K+-dependent depolarization. Based on these data, the criteria for selection as a hit, was a greater or equal to 90% inhibition of the Na+-dependent depolarization and a less than or equal to 20% inhibition of the K+-dependent depolarization. This protocol provided a distinction between compounds that were inert or non-specific in their effects and compounds that specifically block the persistent sodium current.
Optical experiments in microtiter plates were performed on the Voltage/Ion Probe Reader (VIPR) using two 400 nm excitation filters and filter sticks with 460 nm and 570 nm filters on the emission side for the blue and red sensitive PMTs, respectively. The instrument was run in column acquisition mode with 2 or 5 Hz sampling and 30 seconds of recording per column. Starting volumes in each well were 80 mL; usually 240 mL was added to each well during the course of the experiment. The lamp was allowed to warm up for about 20 minutes and power to the PMTs was turned on for about 10 minutes prior to each experiment.
Ratiometric measurements of changes in fluorescent emissions at 460- and 570 nm on the VIPR platform (Aurora Bioscience, San Diego, Calif.) demonstrated that this assay format produces a robust and reproducible fluorescent signal upon depolarization of HEK-Nav 1.3 cells with a Na+/K+ addition. From a normalized ratio of 1.0 in Na+-free media, Na+-dependent depolarization resulted in an increase in the 460/570 ratio to over 2.2 (FIG. 10). Inter-well analysis of the ratios indicated that the amplitude of signal was large enough and consistent enough to be used in high-throughput screening.
Data were analyzed and reported as normalized ratios of intensities measured in the 460 nm and 580 nm channels. The VIPR sampling rate varied between 2 and 5 Hz in different experiments, with 5 Hz used for higher resolution of the peak sodium responses. The process of calculating these ratios was performed as follows. On all plates, column 12 contained TEA-MeSO3 solution with the same DiSBAC2(3) and ESS-AY17 concentrations as used in the cell plates; however no cells were included in column 12. Intensity values at each wavelength were averaged for the duration of the scan. These average values were subtracted from intensity values in all assay wells. The initial ratio obtained from samples 5-10 (Ri) was defined as:




  Ri
  =
  
    
      
        Intensity
        
          
            460
            ⁢
            
              
            
            ⁢
            nm
          
          ,
          
            
          
          ⁢
          
            samples
            ⁢
            
              
            
            ⁢
            5
            ⁢
            
              -
            
            ⁢
            10
          
        
      
      -
      
        background
        
          460
          ⁢
          
            
          
          ⁢
          nm
        
      
    
    
      
        Intensity
        
          
            580
            ⁢
            
              
            
            ⁢
            nm
          
          ,
          
            
          
          ⁢
          
            samples
            ⁢
            
              
            
            ⁢
            5
            ⁢
            
              -
            
            ⁢
            10
          
        
      
      -
      
        background
        
          580
          ⁢
          
            
          
          ⁢
          nm
        
      
    
  




and the ratio obtained from sample f (Rf) was defined as:





  Rf
  =
  
    
      
        Intensity
        
          
            460
            ⁢
            
              
            
            ⁢
            nm
          
          ,
          
            
          
          ⁢
          
            sample
            ⁢
            
              
            
            ⁢
            f
          
        
      
      -
      
        background
        
          460
          ⁢
          
            
          
          ⁢
          nm
        
      
    
    
      
        Intensity
        
          
            580
            ⁢
            
              
            
            ⁢
            nm
          
          ,
          
            sample
            ⁢
            
              
            
            ⁢
            f
          
        
      
      -
      
        background
        
          580
          ⁢
          
            
          
          ⁢
          nm
        
      
    
  




Final data were normalized to the starting ratio of each well and reported as Rf/Ri. The fluorescent response in the Nav 1.3 persistent current assay reached a peak approximately 10 seconds following the start of the run, therefore, the maximum ratio was selected as the readout for the assay (FIG. 10).
The assay format described above allows for quality assurance by measuring both negative (DMSO 0.1%) and positive (tetracaine 10 μM) controls. Every 10th plate in an assay run was a control plate. The data from these plates were used to verify that the assay conditions were optimal and to normalize the data from the test compounds. FIG. 11 shows results from control plates from multiple assays.
In FIG. 11, control plates having wells containing either 0.1% DMSO or 10 μM tetracaine were run after every ninth assay plate. The response to Na+-dependent depolarization was measured and the data were binned into histograms as shown. The mean maximum response (Max) obtained in the presence of (0.1% DMSO) and the mean minimum response (Min) obtained in the presence of 10 μM tetracaine were determined. For quality control, data variance was calculated using a Z′ factor method that compares the difference between the maximum and minimum signals in order to discriminate hit compounds from the background variation, see, e.g., Ji-Hu Zhang et al, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, 4(2) J. Biomol. Screen. 67-73 (1999). This was accomplished by calculating a screening window (z) for each control plate. Data for the run was accepted if 1.0≧Z≧0.5. The Z′ factor is calculated by comparing the difference of the means of a positive and negative control with their respective standard deviations as in equation:




  Z
  =
  
    1
    -
    
      
        
          3
          ⨯
          
            STD
            max
          
        
        +
        
          3
          ⨯
          
            STD
            min
          
        
      
      
        
          Mean
          max
        
        -
        
          Mean
          min
        
      
    
  




Example 2
Screening Assay for Identifying Persistent Sodium Current Blockers Using Single Wavelength Voltage-Sensitive Dyes
To establish an assay plate. HEK-8293 cells stably transfected with a cDNA for the Nav 1.3 sodium channel capable of mediating a persistent sodium current (HEK-Nav 1.3 cells) were grown in Minimum Essential Medium (Invitrogen, Inc., Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen, Inc., Carlsbad, Calif.), 1% Penicillin-Streptomycin (Invitrogen, Inc., Carlsbad, Calif.), 500 mg/mL G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μM TTX (Calbiochem, Inc., San Diego, Calif.) for maintaining selective pressure. Cells were grown in vented cap flasks, in 90% humidity and 10% CO2, to about 80% confluence, harvested by trypsinization and cell density was determined. Approximately 16 to 24 hours before the assay, cells were seeded at 75,000 cells per well in 150 μL of MEM media in clear bottom, black-wall, poly-d-lysine coated 96-well plates (BD Biosciences) and stored in a 5% CO2, 37° C. incubator overnight. This plating procedure resulted in an optimal cell confluence (80%-90%) at the time of the assay.
To examine the ability of test molecules to alter persistent sodium current, the medium was aspirated from the wells and replaced with 40 mL of TEA-MeSO3 (Na+ depletion) solution containing the following: TEA-MeSO3 (140 mM), HEPES-MeSO3 (10 mM), KMeSO3 (4.5 mM), Glucose (10 mM) MgCl2 (1 mM), CaCl2 (1 mM), CdCl2 (0.2 mM) with a pH of 7.4 and an osmolarity of 300-310 mOsm. The Na+ depletion solution also contained 4× of the final test concentration of Molecular Devices membrane potential dye (Molecular Devices Corp., Sunnyvale, Calif.) made up according to manufacturers instructions. The Molecular Devices membrane potential dye is a lipophilic, anionic, bis-oxonol dye that can partition across the cytoplasmic membrane of live cells, dependent on the membrane potential across the plasma membrane. Its fluorescence intensity increases when the dye is bound to cytosolic proteins. When the cells are depolarized, more dye enters the cells, and the increased intracellular concentration of the dye binding to intracellular lipids and proteins causes an increase in fluorescence signal. When the cells are hyperpolarized, dye exits the cells, and the decreased intracellular concentration of dye binding to lipids and proteins results in a decreased of fluorescence signal. The dye was excited at the 488 nm wavelength. At this time either positive or negative control compounds or test molecules were added to the wells of the plate at 1× their final test concentration. The plate was allowed to incubate with the dye and compounds were allowed to incubate for about 25-30 minutes at room temperature in dark.
The assay plates were then transferred to a Fluorometric Imaging Plate Reader (FLIPR-Tetra, Molecular Devices Corp., Sunnyvale, Calif.) for measurement of depolarization induced by addition of the Na+ repletion buffer. The Na+ repletion buffer solution comprised the following: NaMeSO3 (140 mM), HEPES-MeSO3 (10 mM), KMeSO3 (13 mM), Glucose (10 mM) MgCl2 (1 mM), CaCl2 (1 mM), CdCl2 (0.2 mM) with a pH of 7.4 and an osmolarity of 300-310 mOsm. The Na+ depletion solution also contained 1× of the final test concentration of either positive or negative control compounds or test molecules.
The parameters for the FLIPR-Tetra data acquisition were set as follows: the excitation wavelength was set to a bandpass of 510-545 nm; the emission wavelength was set to a bandpass of 565-625 nm; the gain of camera was set between 60-100 with an exposure time of 0.1 s and with an acquisition rate of 5 Hz.
The assay protocol was as follows: after transferring the plates to the FLIPR-Tetra baseline fluorescence was measured for 5 sec at which time 120 μL of the Na+ repletion buffer was added to initiate a depolarizing response (FIG. 14A, Control). This response contained both a specific depolarization a result of Na+ flux across voltage-gated sodium channels (VGSC) and a non-specific depolarization resulting from other Na+-dependent mechanisms in the HEK cells. The background response was revealed in wells that contained an excess of TTX (1 μM) to block all VGSC. Specific responses were measured by subtracting the average response of wells containing 1 μM TTX from the test wells (FIG. 14B).
For data analysis the peak fluorescence amplitude measures from each well of the test plates in the FLIPR were calculated automatically by Screenworks (Molecular Devices Corp, Sunnyvale, Calif.) which determined the maximum peak as the difference from the most positive peak relative to baseline. Peak amplitude measures were then imported to an excel template file which is used to calculate mean and SD. Mean amplitude measures from each drug-treated group were normalized with respect to the mean of control group. Normalized mean amplitude measures from control and drug treated wells were imported into Origin for plotting concentration-response curves of and determination IC50 values of persistent sodium channel blockers.
To test the accuracy and reproducibility of the Na+ depletion/repletion assay on the FLIPR system, two assay formats were used: a screening window format to measure the ability to obtain reproducible data in the single-concentration or HTS mode of screening (FIG. 15A) and a dose-response format to measure the ability to accurately predict IC50 of known reference compounds (FIG. 15B)
Results from 96-well plate setup in the screening window format demonstrated the overall reproducibility of the response (FIG. 16). In this plate Na+ was added to wells A1-H6 to induce depolarization while 1 μM TTX were incubated for 30 minutes in wells A7-H12 prior to Na+ addition. The Na+ channel dependent component of the fluorescence response in wells A7-H12 was blocked by TTX.
Within HTS assays a standard method to evaluate the ability to discriminate hit compounds from the background variation in the signal-to-noise ratio of the assay was calculated using a metric called Z′ factor, see, e.g., Ji-Hu Zhang et al, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, 4(2) J. Biomol. Screen. 67-73 (1999). The Z′ factor was calculated by comparing the difference of the means of a positive and negative control with their respective standard deviations as in equation:




  Z
  =
  
    1
    -
    
      
        
          3
          ⨯
          
            STD
            max
          
        
        +
        
          3
          ⨯
          
            STD
            min
          
        
      
      
        
          Mean
          max
        
        -
        
          Mean
          min
        
      
    
  




Mean Fluorescence peak amplitude and standard deviation from control and drug treated wells was used to calculate the screening window factor (Z′). Peak fluorescence amplitude measures from each well was calculated automatically by Screenworks which determined the maximum peak as the difference from the most positive peak relative to baseline. Peak amplitude measures were then imported to an excel template file which was used to calculate mean and SD. Mean and SD from control and drug treated groups were input to equation 1 for determination of screening window factor (Z′). For the data illustrated in FIG. 15 the Z′ factor was 0.52 (FIG. 16B). Assays were generally considered acceptable when z′ varies between 0.5 and 1.0.
To examine the relative potency of test molecules against persistent sodium currents their IC50 was determined from dose response data as shown in FIG. 17. In this the assay plate in FIG. 17A, wells A1-H2, and A11-H12 received Na+ addition while other wells from column 3-10 were treated with TTX ranging from 0.5 nM to 1000 nM (see plate layout in FIG. 15B). The average fluorescence waveforms from control and TTX treated groups were plotted to illustrate the dose dependent blockade of Na+-induced fluorescence response by TTX (FIG. 17B). Peak amplitude measures from the reduced data were exported to an Excel analysis template file which calculated mean fluorescence amplitude and SD. Mean amplitude measures from test-molecule treated groups were normalized with respect to the mean of control group. Normalized mean amplitude measures from control and TTX treated wells were imported into Origin for determination of IC50. Normalized mean amplitudes were plotted as a function of dose response of TTX in log scale. IC50 was determined by curve fitting using the logistic dose response equation:




  Y
  =
  
    
      
        
          A
          1
        
        -
        
          A
          2
        
      
      
        1
        +
        
          
            (
            
              x
              /
              
                x
                0
              
            
            )
          
          p
        
      
    
    +
    
      A
      2
    
  




where

    
    
        x0=center
        p=power
        A1=Initial Y value
        A2=Final Y value
        The Y value at x0 is half way between the two limiting values A1 and A2 
        Y(x0)=(A1+A2)/2
    
    


Concentration-response curves obtained from three test compounds are shown in FIG. 17C. The IC50 values for TTX, Tetracaine and Lidocaine of 5 nM, 1.8 μM and 32 μM obtained in this assay correspond well with the values obtained using patch-clamp (5 nM, 1 μM and 90 μM respectively).
Example 3
Screening Assay for Identifying Transient Sodium Current Blockers Using Electric Field Stimulation (EFS)
Methods for applying external electric fields to stimulate excitable cells and tissues are well known and have been extensively reviewed, see, e.g., Peter J. Basser and Bradley J. Roth, New Currents in Electrical Stimulation of Excitable Tissues, 2 Annu. Rev. Biomed. Eng. 377-397 (2000); Jaakko Malmivuo and Robert Plonsey, Bioelectromagnetism: Principles and Applications of Bioelectric and Biomagnetic Fields, (Oxford University Press, New York. 472 pp. 1995); and J. Patrick Reilly, Electrical Stimulation and Electropathology, (Cambridge University Press, Cambridge. 1-522 pp. 1992).
To establish an assay to measure the potency of compounds for blocking transient sodium currents in order to compare their potency against blocking persistent sodium currents, HEK-293-Nav 1.3 cells will be grown in Minimum the presence of 500 mg/mL G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μM TTX (Calbiochem, Inc., San Diego, Calif.) to maintain selective pressure.
To measure transient currents cells will be transferred to a recording chamber suitably instrumented with electrodes to produce EFS as described in, e.g., Michael P. Maher & Jesus E. Gonzalez, Multi-well Plate and Electrode Assemblies for Ion Channel Assays, U.S. Pat. No. 6,969,449 (Nov. 29, 2005); Michael P. Maher & Jesus E. Gonzalez, High Throughput Method and System for Screening Candidate Compounds for Activity Against Target Ion Channels, U.S. Pat. No. 6,686,193 (Feb. 3, 2004). and Paul Burnett et al., Electrophysiology Assay Methods, U.S. Patent Publication No. 2004/0115614 (Jun. 17, 2004).
Cells will be loaded with either an appropriate FRET donor/acceptor voltage-sensitive dye pair as described in Example 1 or a single wavelength voltage-sensitive dye as described in Example 2. The cells will be transferred to appropriate device to record membrane potential induced changes in fluorescence, e.g., a VIPR (Aurora Bioscience, San Diego, Calif.) or FLIPR-tetra (Molecular Devices, Sunnyvale, Calif.).
Optical measurement of fluorescent changes in response of EFS will be measured of a series of stimuli. The transient Na+ current produces rapid change in fluorescence due to the rapid depolarization. For quantification of the block of transient current, the amplitude of peak response will be averaged form a series of stimuli. The average response will be converted to activity by normalizing against the difference between the responses in Ringer's solution with DMSO and Ringer's solution containing 10 μM tetracaine or 100 nM TTX. Normalized activity against the transient current will be plotted as a concentration dose response curve and IC50 for block against transient currents can be calculated by a fitting a logistic function to the data.
Example 4
Screening Assay for Identifying Transient Sodium Current Blockers Using Automated Patch-Clamp Technology
HEK-293 cells stably transfected with a cDNA for the Nav 1.3 sodium channel capable of mediating a persistent sodium current (HEK-Nav 1.3 cells) were grown in Minimum Essential Medium (Invitrogen, Inc., Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen, Inc., Carlsbad, Calif.), 1% Penicillin-Streptomycin (Invitrogen, Inc., Carlsbad, Calif.), 500 mg/mL G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μM TTX (Calbiochem, Inc., San Diego, Calif.) for maintaining selective pressure. Cells were grown in vented cap flasks, in 90% humidity and 10% CO2, to about 80% confluence, harvested by trypsinization and cell density was determined. Cells were resuspended at a density of 2×10/mL in the extracelluar solution described below and transferred to either a IonWorks (Molecular Devices, Sunnyvale, Calif.) or Flyscreen (Flyion, GmbH) automated patch clamp for measurement of peak transient Na+ current.
Solutions used for these experiments were as follows: Internal Solution (in mM): 140 KCl, 2 MgCl2 5 EGTA, 10 Hepes pH to 7.2 with KOH; External Solution (in mM): 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 Hepes, 10 Glucose, pH to 7.4 with NaOH.
Na+ currents were elicited by the following pulse protocol: Cells were held at a −90 mV and stimulated every 5 sec with a voltage step to 0 mV for 25 ms. Peak inward current was measured between 1 and 5 ms after the onset of the voltage pulse. After a pre-addition run to determine baseline currents, compounds were applied by and allowed to incubate for 5 minutes and a second reading was then taken to compare currents in the present of either positive or negative control compounds or test compounds. FIG. 18 shows an example of four such experiments from an IonWorks automated patch clamp. The peak currents from the pre-addition run and after exposure to 100 nM TTX are labeled.
To examine the relative potency of test molecules against transient sodium currents their IC50 was determined from dose response data as shown in FIG. 19. Peak current measures from the reduced data shown in FIG. 18 were exported to an Excel analysis template file which calculated mean current amplitude and SD. Mean amplitude measures from test-molecule treated groups were normalized with respect to the mean of control group. Normalized mean amplitude measures from control and TTX treated wells were imported into Origin for determination of IC50. Normalized mean amplitudes were plotted as a function of dose response of TTX in log scale. IC50 was determined by curve fitting using the logistic dose response equation:




  Y
  =
  
    
      
        
          A
          1
        
        -
        
          A
          2
        
      
      
        1
        +
        
          
            (
            
              x
              /
              
                x
                0
              
            
            )
          
          p
        
      
    
    +
    
      A
      2
    
  




where

    
    
        x0=center
        p=power
        A1=Initial Y value
        A2=Final Y value
        The Y value at x0 is half way between the two limiting values A1 and A2 
        Y(x0)=(A1+A2)/2
    
    


IC50 values obtained in this assay were then be compared against IC50 for blocking persistent currents as measured in examples 1 and 2. This allows the calculation of the relative selectivity of block for persistent vs. transient currents.
Example 5
Electrophysiological Assay for Selectivity of Inhibitors of Persistent Sodium Current
To confirm the blocking selectivity of test compounds for persistent sodium current, individual compounds were examined using a whole-cell patch clamp method. HEK cells transfected with Nav 1.3 sodium channels that express transient and persistent sodium currents were plated onto glass coverslips and cultured in MEM cell culture media with Earle's salts and GlutaMAX (Invitrogen, Inc., Carlsbad, Calif.) supplemented with: 10% Fetal bovine serum, heat inactivated (Invitrogen, Inc., Carlsbad, Calif.), 0.1 mM MEM non-essential amino acids (Invitrogen, Inc., Carlsbad, Calif.), 10 mM HEPES (Invitrogen, Inc., Carlsbad, Calif.), 1% Penicillin/Streptomycin (Invitrogen, Inc., Carlsbad, Calif.).
After an incubation period of from 24 to 48 hours the culture medium was removed and replaced with external recording solution (see below). Whole cell patch clamp experiments were performed using an EPC10 amplifier (HEKA Instruments, Lambrecht, Germany.) linked to an IBM compatible personal computer equipped with PULSE software. Borosilicate glass patch pipettes were pulled to a fine tip on a P90 pipette puller (Sutter Instrument Co., Novato, Calif.) and were polished (Microforge, Narishige, Japan) to a resistance of about 1.5 Mohm when filled with intracellular recording solution (Table 3).
Persistent and transient currents in HEK cells expressing Nav 1.3 channels were measured by applying 200-msec depolarizations from a holding potential of −90 mV to 0 mV. Background currents that remained in the presence of 500 nM TTX were subtracted from all traces. Drugs were perfused directly into the vicinity of the cells using a microperfusion system.







TABLE 3







Patch Clamp Solutions








External Recording Solution
Internal Recording Solution










Compound
Concentration
Compound
Concentration















NaCl
127
mM
CsMeSO3
125
mM


HEPES (free acid)
10
mM
CsCl
25
mM


KCl
5
mM
NaHEPES
10
mM


CsCl
5
mM
Amphotericin
240
μg/mL


Glucose
10
mM


MgCl2
0.6
mM


CaCl2
1.2
mM


CdCl2
200
μM








pH to 7.4 with NaOH @ room temp.
pH 7.20 with CsOH300 mOsm


290 mOsm.









Under control conditions, depolarizing pulses elicited a large transient inward current that declined to a smaller persistent current, which remained stable during the remainder of the pulse (FIG. 12, control). Addition of 500 nM TTX completely blocked both the transient and persistent currents (FIG. 12, TTX). Application of 3 μM of Compound 1 produced a much different effect. Inspection of FIG. 12 reveals that the Compound 1 blocked 99% of the persistent current while only reducing the transient current by 16%. Dose-response analysis for Compound 1 demonstrates its significant selectivity for blocking the persistent sodium current relative to the transient sodium current over a four order of magnitude range (FIG. 13).

## Claims

1. A method for identifying a selective blocker of a persistent Na+ channel whereby the method comprises the steps of:
a) providing a test sample 1 comprising
i) a Na+-free physiological solution;
ii) a fast-response voltage-sensitive fluorescence dye;
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; and
iv) a potential Na+ channel blocker;

b) depolarizing membrane of the cell in the test sample 1;
c) generating a current through the persistent Na+ channel by adding Na+ to test sample 1 at least 10 msec after step (b);
d) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1;
e) providing a control sample 1 comprising
i) a Na+-free physiological solution;
ii) a fast-response voltage-sensitive fluorescence dye; and
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel;

f) depolarizing membrane of the cell in the control sample 1;
g) generating a current through the persistent Na+ channel by adding Na+ ions to the control sample 1 at least 10 msec after step (f);
h) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1;
i) determining the relative emitted fluorescence 1 by comparing the emitted fluorescence from step (d) to the emitted fluorescence from step (h);
j) providing a test sample 2 comprising
i) a physiological solution;
ii) a fast-response voltage-sensitive fluorescence dye;
iii) a cell having a K+ channel and a transient Na+ channel; and
iv) a potential Na+ channel blocker

k) depolarizing membrane of the cell in test sample 2;
l) detecting the fluorescence emitted by the voltage-sensitive dye in test sample 2;
m) providing a control sample 2 comprising
i) a physiological solution;
ii) a fast-response voltage-sensitive fluorescence dye; and
iii) a cell having a K+ channel and a transient Na+ channel;

n) depolarizing membrane of the cell in control sample 2;
o) detecting the fluorescence emitted by the voltage-sensitive dye in control sample 2;
p) determining a relative emitted fluorescence 2 by comparing the emitted fluorescence from step (l) to the emitted fluorescence from step (o);
q) comparing the relative emitted fluorescence 1 in step (i) with the relative emitted fluorescence 2 in step (p), wherein a decrease is emitted in emitted fluorescence 1 relative to emitted fluorescence 2 is indicative of the presence of a selective persistent Na+ channel blocker in the test sample.

2. The method according to claim 1, wherein the fast-response voltage-sensitive fluorescence dye is selected from the group consisting of di-2-ANEPEQ (JPW1114), di-1-ANEPIA, di-8-ANEPPQ, di-12-ANEPPQ, di-4-ANEPPS, di-8-ANEPPS, di-18:2-ANEPPS, RGA-30, RH-155, RH-795, RH-237, RH-421, RH-414 and WW 781.

3. The method according to claim 1, wherein the cell expresses a persistent Na+ channel selected from the group consisting of Nav 1.3, Nav 1.5, Nav 1.6 and Nav 1.9.

4. The method according to claim 1, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a single fluorescent dye method.

5. The method according to claim 1, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a FRET method.

6. A method for identifying a blocker of a persistent Na+ channel whereby the method comprises the steps of:
a) providing a test sample 1 comprising
i) a Na+-free physiological solution;
ii) a fast-response voltage-sensitive fluorescence dye;
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; and
iv) a potential Na+ channel blocker;

b) depolarizing membrane of the cell in the test sample 1;
c) generating a current through the persistent Na+ channel by adding Na+ to test sample 1 at least 10 msec after step (b);
d) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1;
e) providing a control sample 1 comprising
i) a Na+-free physiological solution;
ii) a fast-response voltage-sensitive fluorescence dye; and
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel;

f) depolarizing membrane of the cell in the control sample 1;
g) generating a current through the persistent Na+ channel by adding Na+ ions to the control sample 1 at least 10 msec after step (f);
h) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1;
i) comparing the emitted fluorescence from step (d) to the emitted fluorescence from step (h), wherein a decrease in emitted fluorescence from a test sample relative to a control sample is indicative of the presence of a persistent Na+ channel blocker in the test sample.

7. The method according to claim 6, wherein the fast-response voltage-sensitive fluorescence dye is selected from the group consisting of di-2-ANEPEQ (JPW1114), di-1-ANEPIA, di-8-ANEPPQ, di-12-ANEPPQ, di-4-ANEPPS, di-8-ANEPPS, di-18:2-ANEPPS, RGA-30, RH-155, RH-795, RH-237, RH-421, RH-414 and WW 781.

8. The method according to claim 6, wherein the cell expresses a persistent Na+ channel selected from the group consisting of Nav 1.3, Nav 1.5, Nav 1.6 and Nav 1.9.

9. The method according to claim 6, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a single fluorescent dye method.

10. The method according to claim 6, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a FRET method.

11. A method for identifying a selective blocker of a persistent Na+ channel whereby the method comprises the steps of:
a) providing a test sample 1 comprising
i) a Na+-free physiological solution;
ii) a slow-response voltage-sensitive fluorescence dye;
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; and
iv) a potential Na+ channel blocker;

b) depolarizing membrane of the cell in the test sample 1;
c) generating a current through the persistent Na+ channel by adding Na+ to test sample 1 at least 10 msec after step (b);

d) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1;
e) providing a control sample 1 comprising
i) a Na+-free physiological solution;
ii) a slow-response voltage-sensitive fluorescence dye; and
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel;

f) depolarizing membrane of the cell in the control sample 1;
g) generating a current through the persistent Na+ channel by adding Na+ ions to the control sample 1 at least 10 msec after step (f);
h) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1;
i) determining the relative emitted fluorescence 1 by comparing the emitted fluorescence from step (d) to the emitted fluorescence from step (h);
j) providing a test sample 2 comprising
i) a physiological solution;
ii) a slow-response voltage-sensitive fluorescence dye;
iii) a cell having a K+ channel and a transient Na+ channel; and
iv) a potential Na+ channel blocker

k) depolarizing membrane of the cell in test sample 2;
l) detecting the fluorescence emitted by the voltage-sensitive dye in test sample 2;
m) providing a control sample 2 comprising
i) a physiological solution;
ii) a slow-response voltage-sensitive fluorescence dye; and
iii) a cell having a K+ channel and a transient Na+ channel;

n) depolarizing membrane of the cell in control sample 2;
o) detecting the fluorescence emitted by the voltage-sensitive dye in control sample 2;
p) determining a relative emitted fluorescence 2 by comparing the emitted fluorescence from step (l) to the emitted fluorescence from step (o);
q) comparing the relative emitted fluorescence 1 in step (i) with the relative emitted fluorescence 2 in step (p), wherein a decrease in emitted fluorescence 1 relative to emitted fluorescence 2 is indicative of the presence of a selective persistent Na+ channel blocker in the test sample.

12. The method according to claim 11, wherein the slow-response voltage-sensitive fluorescence dye is selected from the group consisting of DiSBAC4(3), DiBAC4(5), DiBAC4(3), DiOC5(3), DiOC6(3), DiSC3(5), DiOC2(3), DiNOC1 (3), DilC1(5), merocyanine 540, Oxonol V, Oxonol VI, rhodamine 123, TMRM, TMRE and CBlC2(3).

13. The method according to claim 11, wherein the cell expresses a persistent Na+ channel selected from the group consisting of Nav 1.3, Nav 1.5, Nav 1.6 and Nav 1.9.

14. The method according to claim 11, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a single fluorescent dye method.

15. The method according to claim 11, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a FRET method.

16. A method for identifying a blocker of a persistent Na+ channel whereby the method comprises the steps of:
a) providing a test sample 1 comprising
i) a Na+-free physiological solution;
ii) a slow-response voltage-sensitive fluorescence dye;
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel; and
iv) a potential Na+ channel blocker;

b) depolarizing membrane of the cell in the test sample 1;
c) generating a current through the persistent Na+ channel by adding Na+ to test sample 1 at least 10 msec after step (b);
d) detecting fluorescence emitted by the voltage-sensitive dye in test sample 1;
e) providing a control sample 1 comprising
i) a Na+-free physiological solution;
ii) a slow-response voltage-sensitive fluorescence dye; and
iii) a cell having a K+ channel, a transient Na+ channel and a persistent Na+ channel;

f) depolarizing membrane of the cell in the control sample 1;
g) generating a current through the persistent Na+ channel by adding Na+ ions to the control sample 1 at least 10 msec after step (f);
h) detecting fluorescence emitted by the voltage-sensitive dye in the control sample 1;
i) comparing the emitted fluorescence from step (d) to the emitted fluorescence from step (h), wherein a decrease in emitted fluorescence from a test sample relative to a control sample is indicative of the presence of a persistent Na+ channel blocker in the test sample.

17. The method according to claim 16, wherein the slow-response voltage-sensitive fluorescence dye is selected from the group consisting of DiSBAC4(3), DiBAC4(5), DiBAC4(3), DiOC5(3), DiOC6(3), DiSC3(5), DiOC2(3), DiNOC1 (3), DilC1(5), merocyanine 540, Oxonol V, Oxonol VI, rhodamine 123, TMRM, TMRE and CBlC2(3).

18. The method according to claim 16, wherein the cell expresses a persistent Na+ channel selected from the group consisting of Nav 1.3, Nav 1.5, Nav 1.6 and Nav 1.9.

19. The method according to claim 16, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a single fluorescent dye method.

20. The method according to claim 16, wherein the emitted fluorescence from step (d), (h), (l) or (o) is detected by a FRET method.