bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature dependence of Na currents in rabbit and frog muscle membranes.
Authors: Kirsch G E , Sykes J S
Published: May 2004

The effect of temperature (0-22 degrees C) on the kinetics of Na channel conductance was determined in voltage-clamped rabbit and frog skeletal muscle fibers using the triple-Vaseline-gap technique. The Hodgkin-Huxley model was used to extract kinetic parameters; the time course of the conductance change during step depolarization followed m3h kinetics. Arrhenius plots of activation time constants (tau m), determined at both moderate (-10 to -20 mV) and high (+100 mV) depolarizations, were linear in both types of muscle. In rabbit muscle, Arrhenius plots of the inactivation time constant (tau h) were markedly nonlinear at +100 mV, but much less so at -20 mV. The reverse situation was found in frog muscle. The contrast between the highly nonlinear Arrhenius plot of tau h at +100 mV in rabbit muscle, compared with that of frog muscle, was interpreted as revealing an intrinsic nonlinearity in the temperature dependence of mammalian muscle Na inactivation. These results are consistent with the notion that mammalian cell membranes undergo thermotropic membrane phase transitions that alter lipid-channel interactions in the 0-22 degrees C range. Furthermore, the observation that Na channel activation appears to be resistant to this effect suggests that the gating mechanisms that govern activation and inactivation reside in physically distinct regions of the channel.
Gating of Na channels. Inactivation modifiers discriminate among models.
Authors: Gonoi T , Hille B
Published: May 2004

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.
Dynamics of turtle cones.
Authors: Naka K I , Itoh M A , Chappell R L
Published: May 2004

The response dynamics of turtle photoreceptors (cones) were studied by the cross-correlation method using a white-noise-modulated light stimulus. Incremental responses were characterized by the kernels. White-noise-evoked responses with a peak-to-peak excursion of greater than 5 mV were linear, with mean square errors of approximately 8%, a degree of linearity comparable to the horizontal cell responses. Both a spot (0.17 mm diam) and a large field of light produced almost identical kernels. The amplitudes of receptor kernels obtained at various mean irradiances fitted approximately the Weber-Fechner relationship and the mean levels controlled both the amplitude and the response dynamics; kernels were slow and monophasic at low mean irradiance and were fast and biphasic at high mean irradiance. This is a parametric change and is a piecewise linearization. Horizontal cell kernels evoked by the small spot of light were monophasic and slower than the receptor kernels produced by the same stimulus. Larger spots of light or a steady annular illumination transformed the slow horizontal cell kernel into a fast kernel similar to those of the receptors. The slowing down of the kernel waveform was modeled by a simple low-pass circuit and the presumed feedback from horizontal cells onto cones did not appear to play a major role.
Two types of potassium channels in murine T lymphocytes.
Authors: Decoursey T E , Chandy K G , Gupta S , Cahalan M D
Published: May 2004

The properties of two types of K+ channels in murine T lymphocytes are described on the basis of whole-cell and isolated-patch recordings using the gigohm-seal technique. Type l (standing for "lpr gene locus" or "large") channels were characterized mainly in T cells from mutant MRL/MpJ-lpr/lpr mice, in which they are present in large numbers. Type n ("normal") K+ channels are abundant and therefore most readily studied in concanavalin A-activated T cells from four strains of mice, MRL-+/+, CBA/J, C57BL/6J, and BALB/c. Type l channels, compared with type n, are activated at potentials approximately 30 mV more positive, and close much more rapidly upon repolarization. Type l channels inactivate more slowly and less completely than type n during maintained depolarization, but recover from inactivation more rapidly, so that little inactivation accumulates during repetitive pulses. Type l channels have a higher unitary conductance (21 pS) than type n (12 pS) and are less sensitive to block by external Co++, but are 100-fold more sensitive to block by external tetraethylammonium (TEA), with half-block of type l channels at 50-100 microM TEA compared with 8-16 mM for type n. TEA blocks both types of channels by reducing the apparent single channel current amplitude, with a dose-response relation similar to that for blocking macroscopic currents. Murine type n K+ channels resemble K+ channels in human T cells.
Convective paracellular solute flux. A source of ion-ion interaction in the epithelial transport equations.
Authors: Weinstein A M
Published: May 2004

An electrolyte model of an epithelium (a cell and a tight junction in parallel, both in series with a lateral interspace basement membrane) is analyzed using the formalism of nonequilibrium thermodynamics. It is shown that if the parallel structures are heteroporous (i.e., reflection coefficients for two ion species differ between the components), then a cross-term will appear in the overall transport equations of the epithelium. Formally, this cross-term represents an ion-ion interaction. With respect to the rat proximal tubule, data indicating epithelial ionic reflection coefficients less than unity, together with the assumption of no transcellular solvent drag, imply the presence of convective paracellular solute flux. This means that a model applicable to a heteroporous structure must be used to represent the tubule, and, in particular, the cross-terms for ion-ion interaction must also be evaluated in permeability determinations. A series of calculations is presented that permits the estimation of the Na-Cl interaction for rat proximal tubule from available experimental data. One consequence of tubule heteroporosity is that an electrical potential may be substantially less effective than an equivalent concentration gradient in driving reabsorptive ion fluxes.
Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes.
Authors: Alpern R J , Chambers M
Published: May 2004

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.
Circadian rhythms in the green sunfish retina.
Authors: Dearry A , Barlow R B
Published: May 2004

We investigated the occurrence of circadian rhythms in retinomotor movements and retinal sensitivity in the green sunfish, Lepomis cyanellus. When green sunfish were kept in constant darkness, cone photoreceptors exhibited circadian retinomotor movements; rod photoreceptors and retinal pigment epithelium (RPE) pigment granules did not. Cones elongated during subjective night and contracted during subjective day. These results corroborate those of Burnside and Ackland (1984. Investigative Ophthalmology and Visual Science. 25:539-545). Electroretinograms (ERGs) recorded in constant darkness in response to dim flashes (lambda = 640 nm) exhibited a greater amplitude during subjective night than during subjective day. The nighttime increase in the ERG amplitude corresponded to a 3-10-fold increase in retinal sensitivity. The rhythmic changes in the ERG amplitude continued in constant darkness with a period of approximately 24 h, which indicates that the rhythm is generated by a circadian oscillator. The spectral sensitivity of the ERG recorded in constant darkness suggests that cones contribute to retinal responses during both day and night. Thus, the elongation of cone myoids during the night does not abolish the response of the cones. To examine the role of retinal efferents in generating retinal circadian rhythms, we cut the optic nerve. This procedure did not abolish the rhythms of retinomotor movement or of the ERG amplitude, but it did reduce the magnitude of the nighttime phases of both rhythms. Our results suggest that more than one endogenous oscillator regulates the retinal circadian rhythms in green sunfish. Circadian signals controlling the rhythms may be either generated within the eye or transferred to the eye via a humoral pathway.
Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block.
Authors: Green W N , Weiss L B , Andersen O S
Published: May 2004

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.
ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants.
Authors: Schoenberg M , Eisenberg E
Published: May 2004

We have studied the binding of adenosine diphosphate (ADP) to attached cross-bridges in chemically skinned rabbit psoas muscle fibers and the effect of that binding on the cross-bridge detachment rate constants. Cross-bridges with ADP bound to the active site behave very similarly to cross-bridges without any nucleotide at the active site. First, fiber stiffness is the same as in rigor, which presumably implies that, as in rigor, all the cross-bridges are attached. Second, the cross-bridge detachment rate constants in the presence of ADP, measured from the rate of decay of the force induced by a small stretch, are, over a time scale of minutes, similar to those seen in rigor. Because ADP binding to the active site does not cause an increase in the cross-bridge detachment rate constants, whereas binding of nucleotide analogues such as adenyl-5'-yl imidodiphosphate (AMP-PNP) and pyrophosphate (PPi) do, it was possible, by using ADP as a competitive inhibitor of PPi or AMP-PNP, to measure the competitive inhibition constant and thereby the dissociation constant for ADP binding to attached cross-bridges. We found that adding 175 microM ADP to 4 mM PPi or 4 mM AMP-PNP produces as much of a decrease in the apparent cross-bridge detachment rate constants as reducing the analogue concentration from 4 to 1 mM. This suggests that ADP is binding to attached cross-bridges with a dissociation constant of approximately 60 microM. This value is quite similar to that reported for ADP binding to actomyosin subfragment-1 (acto-S1) in solution, which provides further support for the idea that nucleotides and nucleotide analogues seem to bind about as strongly to attached cross-bridges in fibers as to acto-S1 in solution (Johnson, R.E., and P. H. Adams. 1984. FEBS Letters. 174:11-14; Schoenberg, M., and E. Eisenberg. 1985. Biophysical Journal. 48:863-871; Biosca, J.A., L.E. Greene, and E. Eisenberg. 1986. Journal of Biological Chemistry. 261:9793-9800).
Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers.
Authors: Kenyon J L , Sutko J L
Published: May 2004

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.
Monovalent and divalent cation permeation in acetylcholine receptor channels. Ion transport related to structure.
Authors: Dani J A , Eisenman G
Published: May 2004

Single channel patch-clamp techniques were used to study nicotinic acetylcholine receptors in cultured rat myotubes. The single channel conductance in pure cesium and sodium levels off at high concentrations, as if a binding site within the channel were saturating. The conductances at very low concentrations, however, are larger than predicted by the simplest one-site transport model fitted to the high-concentration data. At low concentrations, the current-voltage relations are inwardly rectifying, but they become more ohmic if a small amount of divalent cations is added externally. Magnesium and barium are good permeants that have rather high affinities for the channel. Upon adding low millimolar concentrations of these divalent cations externally to a membrane bathed in pure cesium, the inward current carried by cesium is decreased. As more divalent cations are added, the inward-going currents continued to decrease and the divalent cation replaces cesium as the main current carrier. The ion transport data are described by considering the size, shape, and possible net charge of the channel. In that way, even the complex features of transport are explained in a realistic physical framework. The results are consistent with the channel having long, wide, multiply occupied vestibules that serve as transition zones to the short, selective, singly occupied narrow region of the channel. A small amount of net negative charge within the pore could produce concentration-dependent potentials that provide a simple explanation for the more complicated aspects of the permeation properties.
A comparison of calcium-activated potassium channel currents in cell-attached and excised patches.
Authors: Pallotta B S , Hepler J R , Oglesby S A , Harden T K
Published: May 2004

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.
Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.
Authors: Hermann A , Erxleben C
Published: May 2004

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.
Cyclic AMP inhibits Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder epithelium.
Authors: Reuss L
Published: May 2004

Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on apical membrane Cl-/HCO3- exchange and electrodiffusive HCO3- transport in Necturus gallbladder epithelium. Intracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophylline (3 X 10(-3) M) or the adenylate cyclase activator forskolin (10(-5) M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H+ and HCO3-. When the same experiment is performed after addition of amiloride or removal of Na+ from the mucosal bathing medium, alkalinization is observed since H+ transport is either inhibited or reversed, whereas HCO3- secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular Cl- activity (aCli) upon reducing mucosal solution [Cl-] to either 10 or 0 mM, although membrane voltage and resistance measurements were consistent with stimulation of apical membrane electrodiffusive Cl- permeability. Estimates of the conductive flux, obtained by either reducing simultaneously mucosal [Cl-] and [HCO3-] or lowering [Cl-] alone in the presence of a blocker of anion exchange (diphenylamine-2-carboxylate), indicate that elevation of intracellular cAMP inhibited the anion exchanger by approximately 50%. Measurements of net Cl- uptake upon increasing mucosal Cl- from nominally zero to levels ranging from 2.5 to 100 mM suggest that the mechanism of inhibition is a decrease in Vmax. Consistent with these results, the rate of intracellular alkalinization upon reducing external Cl- was also inhibited significantly by theophylline. Reducing mucosal solution [HCO3-] from 10 to 1 mM under control conditions caused intracellular acidification and an increase in aCli. Theophylline inhibited both changes, by 62 and 32%, respectively. These data indicate that elevation of intracellular cAMP inhibits apical membrane anion (Cl-/HCO3-) exchange. Studies of the effects of rapid changes in mucosal [HCO3-] on membrane voltages and the apparent ratio of membrane resistances, both in the presence and in the absence of theophylline, with or without Cl- in the mucosal solution, do not support the hypothesis that cAMP produces a sizable increase in apical membrane electrodiffusive HCO3- permeability.
Activation of electroneutral K flux in Amphiuma red blood cells by N-ethylmaleimide. Distinction between K/H exchange and KCl cotransport.
Authors: Adorante J S , Cala P M
Published: May 2004

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.
The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin.
Authors: Huang L Y , Yatani A , Brown A M
Published: May 2004

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.
Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel.
Authors: Miller C , Latorre R , Reisin I
Published: May 2004

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.
Mechanisms contributing to the cardiac inotropic effect of Na pump inhibition and reduction of extracellular Na.
Authors: Bers D M
Published: May 2004

Reduction of the transsarcolemmal [Na] gradient in rabbit cardiac muscle leads to an increase in the force of contraction. This has frequently been attributed to alteration of Ca movements via the sarcolemmal Na/Ca exchange system. However, the specific mechanisms that mediate the increased force at individual contractions have not been clearly established. In the present study, the [Na] gradient was decreased by reduction of extracellular [Na] or inhibition of the Na pump by either the cardioactive steroid acetylstrophanthidin or by reduction of extracellular [K]. Contractile performance and changes in extracellular Ca (sensed by double-barreled Ca-selective microelectrodes) were studied in order to elucidate the underlying basis for the increase in force. In the presence of agents that inhibit sarcoplasmic reticulum (SR) function (10 mM caffeine, 100-500 nM ryanodine), reduction of the [Na] gradient produced increases in contractile force similar to that observed in the absence of caffeine or ryanodine. It is concluded that an intact, functioning SR is not required for the inotropic effect of [Na] gradient reduction (at least in rabbit ventricle). However, this does not exclude a possible contribution of enhanced SR Ca release in the inotropic response to [Na] gradient reduction in the absence of caffeine or ryanodine. Acetylstrophanthidin (3-5 microM) usually leads to an increase in the magnitude of extracellular Ca depletions associated with individual contractions. However, acetylstrophanthidin can also increase extracellular Ca accumulation during the contraction, especially at potentiated contractions. This extracellular Ca accumulation can be suppressed by ryanodine and it is suggested that this apparent enhancement of Ca efflux is secondary to an enhanced release of Ca from the SR. Under conditions where Ca efflux during contractions is minimized (after a rest interval in the presence of ryanodine), acetylstrophanthidin increased both the rate and the extent of extracellular Ca depletions. Thus, acetylstrophanthidin can increase both Ca influx and Ca efflux during the cardiac muscle contraction. These results can be explained by a simple model where the direction of net Ca flux via Na/Ca exchange during the action potential is determined by the changes in reversal potential of the Na/Ca exchange. Reduction of the [Na] gradient may well lead to net cellular Ca uptake (via Na/Ca exchange) and may also elevate the resting intracellular [Ca].(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of cyclic GMP on the kinetics of the photocurrent in rods and in detached rod outer segments.
Authors: Hestrin S , Korenbrot J I
Published: May 2004

We investigated the effects of high concentrations of cytoplasmic cyclic GMP on the photocurrent kinetics and light sensitivity of the tiger salamander rod both in intact cells and in detached outer segments. Photoreceptors were internally perfused with cGMP by applying patch pipettes containing cGMP to the inner or outer segment. Large increases in the concentration of cGMP in the outer segment cytoplasm were achieved only when the patch pipette was applied directly to the outer segment. The dark-current amplitude increased with increasing cGMP concentrations up to approximately 1,400 pA. Internal perfusion with 5.0 mM cGMP introduced a delay of 1-3 s in the photocurrent. The magnitude of the delay was inversely proportional to the light intensity. In addition, the photocurrent time course was slowed down and the light sensitivity, measured 1 s after the flash, was decreased approximately 100-fold when compared with that of the intact cell. The observed effects of cGMP were compared with those predicted by a model that assumes that the initial photocurrent time course is determined by the kinetics of the light-activated phosphodiesterase (PDE) and the cGMP dependence of the light-sensitive channels. At high concentrations of cGMP, the experimental data were similar to those predicted by the model and based on the known biochemical properties of the light-activated PDE and cGMP-activated channels.
Equilibrium properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.
Authors: Yoshii K , Yu L , Mayne K M , Davidson N , Lester H A
Published: May 2004

This study used messenger RNA encoding each subunit (alpha, beta, gamma and delta) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of alpha, beta, gamma, and delta were injected into oocytes. After allowing 2-3 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [125I]alpha-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (greater than 1,000-fold range at 1 microM). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of alpha-bungarotoxin-binding sites. Five combinations were tested for d-tubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 microM. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the beta subunit on voltage sensitivity of the response: gACh(-90 mV)/gACh(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if beta M replaces beta T. Also, combinations including gamma T or delta M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 microS/fmol at -60 mV; EACh is consistently lower for alpha M. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.
Nontransducing rhodopsin.
Authors: Levine E , Crain E , Robinson P , Lisman J
Published: May 2004

Rhodopsin is converted by light to an active photoproduct that triggers the transduction cascade. The active photoproduct must then be inactivated by some kind of chemical modification. The question addressed here is whether photoconversion of the inactive photoproduct to rhodopsin creates a modified form of rhodopsin that is unable to support transduction. This question was investigated in ultraviolet receptors of Limulus median eye by measuring the relative quantum efficiency of excitation after photoregeneration of rhodopsin from the inactive photoproduct. The results show that when this newly created rhodopsin absorbs a photon, no receptor potential is generated; i.e., the pigment is nontransducing. A dark process requiring 30-60 min returns rhodopsin to its transducing form.
A derivative of amiloride blocks both the light-regulated and cyclic GMP-regulated conductances in rod photoreceptors.
Authors: Nicol G D , Schnetkamp P P , Saimi Y , Cragoe E J , Bownds M D
Published: May 2004

Vertebrate rod photoreceptors in the dark maintain an inward current across the outer segment membrane. The photoresponse results from a light-induced suppression of this dark current. The light-regulated current is not sensitive to either tetrodotoxin or amiloride, potent blockers of Na+ channels. Here, we report that a derivative of amiloride, 3',4'-dichlorobenzamil (DCPA), completely suppresses the dark current and light response recorded from rod photoreceptors. DCPA also blocks a cyclic GMP-activated current in excised patches of rod plasma membrane and a cGMP-induced Ca++ flux from rod disk membranes. These results are consistent with the notion that the Ca++ flux mechanism in the disk membrane and the light-regulated conductance in the plasma membrane are identical. DCPA also inhibits the Na/Ca exchange mechanism in intact rods, but at a 5-10-fold-higher concentration than is required to block the cGMP-activated flux and current. The blocking action of DCPA in 10 nM Ca++ is different from that in 1 mM Ca++, which suggests either that the conductance state of the light-regulated channel may be modified in high and low concentrations of Ca++, or that there may be two ionic channels in the rod outer segment membrane.
Passive properties and membrane currents of canine ventricular myocytes.
Authors: Tseng G N , Robinson R B , Hoffman B F
Published: May 2004

The membrane potential and membrane currents of single canine ventricular myocytes were studied using either single microelectrodes or suction pipettes. The myocytes displayed passive membrane properties and an action potential configuration similar to those described for multicellular dog ventricular tissue. As for other cardiac cells, in canine ventricular myocytes: (a) an inward rectifier current plays an important role in determining the resting membrane potential and repolarization rate; (b) a tetrodotoxin-sensitive Na current helps maintain the action potential plateau; and (c) the Ca current has fast kinetics and a large amplitude. Unexpected findings were the following: (a) in approximately half of the myocytes, there is a transient outward current composed of two components, one blocked by 4-aminopyridine and the other by Mn or caffeine; (b) there is clearly a time-dependent outward current (delayed rectifier current) that contributes to repolarization; and (c) the relationship of maximum upstroke velocity of phase 0 to membrane potential is more positive and steeper than that observed in cardiac tissues from Purkinje fibers.
Two ATP-activated conductances in bullfrog atrial cells.
Authors: Friel D D , Bean B P
Published: May 2004

Currents activated by extracellular ATP were studied in single voltage-clamped bullfrog atrial cells. Rapid application of ATP elicited currents carried through two different conductance pathways: a rapidly desensitizing conductance reversing near -10 mV, and a maintained, inwardly rectifying conductance reversing near -85 mV. ATP activated the desensitizing component of current with a K 1/2 of approximately 50 microM and the maintained component with a K 1/2 of approximately 10 microM. Both types of current were activated by ATP but not by adenosine, AMP, or ADP. The desensitizing current was selectively inhibited by alpha, beta-methylene ATP, and the maintained, inwardly rectifying current was selectively suppressed by extracellular Cs. The desensitizing component of current was greatly reduced when extracellular Na was replaced by N-methylglucamine, but was slightly augmented when Na was replaced by Cs. GTP, ITP, and UTP were all ineffective in activating the desensitizing current, and of a variety of ATP analogues, only ATP-gamma-S was effective. Addition of EGTA or BAPTA to the intracellular solution did not obviously affect the desensitizing current. Fluctuation analysis of currents through the desensitizing conductance suggested that current is carried through ionic channels with a small (less than pS) unitary conductance.
Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials.
Authors: Rorsman P , Hellman B
Published: May 2004

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage-activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5-20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.
Two types of Ca2+ currents with different sensitivities to organic Ca2+ channel antagonists in guinea pig pancreatic alpha 2 cells.
Authors: Rorsman P
Published: May 2004

The possibility that guinea pig pancreatic alpha 2 cells are equipped with more than one type of Ca2+ channel was explored using the patch-electrode voltage-clamp technique. At a holding potential of -100 mV, a slowly developing (tau m approximately 5 ms at -40 mV assuming m2 kinetics) Ca2+ current appeared. This conductance first became detectable at potentials of about -60 mV and reached a maximum amplitude of 50-100 pA between -30 and -20 mV. During long depolarizations, it inactivated completely (tau h approximately 100 ms at -40 mV). Half-maximal steady state inactivation was observed at about -60 mV. A second, more rapidly developing (tau m approximately 2 ms at 0 mV) Ca2+ current was observed during pulses to -40 mV and above. It had a peak amplitude of 150-200 pA between 0 and 10 mV, was less dependent on the holding potential, and inactivated very little, even during long pulses. Both conductances were blocked by Co2+ but were unaffected by tetrodotoxin. The rapidly developing current differed from the slowly developing one in being sensitive to the antagonists D-600 and nifedipine, conducting Ba2+ better than Ca2+, increasing upon exposure to forskolin, and showing time-dependent decay (rundown). These findings indicate that the alpha 2 cells are equipped with two kinds of Ca2+ channels.
The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain.
Authors: Nachshen D A , Drapeau P
Published: May 2004

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.