bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic responses of electrically coupled systems.
Authors: Publicover N G
Published: May 2004

An identified pair of electrically coupled neurons in the buccal ganglion of the freshwater snail Helisoma trivolvis is an experimentally accessible model of electrical synaptic transmission. In this investigation, electrical synaptic transmission is characterized using sinusoidal frequency (Bode) responses computed by Laplace transforms and responses to brief stimuli. The frequency response of the injected neuron shows a 20-dB/decade attenuation and a phase shift from 0 degree at low frequencies to -90 degrees at high frequencies. The response of a coupled cell shows a 40-dB/decade attenuation and a phase shift from 0 degrees at low frequencies to -180 degrees at high frequencies. A simple mathematical model of electrical synaptic transmission is described that displays each of these crucial features of the measured frequency responses. Methods are described to estimate the frequency responses of coupled systems based on presynaptic measurements. The responses of the coupled system to brief pulses of current were computed using the principle of superposition. The electrical properties of coupled systems impose a minimum delay in reaching a peak in all postsynaptic responses. The delays in the postsynaptic responses to brief stimuli are related to the electrical and anatomical parameters of coupled networks.
A potassium-proton symport in Neurospora crassa.
Authors: Rodriguez-Navarro A , Blatt M R , Slayman C L
Published: May 2004

Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1-10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants.
Destruction and reorganization of the receptor membrane in labellar chemosensory cells of the blowfly. Recovery of responses to sugar after destruction.
Authors: Ninomiya M , Ozaki M , Kashihara Y , Morita H
Published: May 2004

Recovery from destruction by sodium deoxycholate (DOC) was studied with the receptor membrane of the blowfly, Phormia regina. The recovery can be divided into two processes, colchicine dependent and colchicine independent. The colchicine-dependent process was completely depressed by pretreatment with colchicine at 5 mM for 2 min (partially at 0.1 mM for 10 min), but the colchicine-independent one persisted. Vinblastine also caused depression but lumicolchicine did not. Records of responses obtained from the DOC-treated sugar receptor showed long response latencies that gradually became indistinct with recovery. Colchicine also affected this change in response latency after the DOC treatment. These results suggest that the colchicine-dependent recovery process is related to microtubules in the distal process of the receptor cell. The recovery time course and the change in response latency could be quantitatively explained by the simple assumptions that DOC underwent desorption from the receptor membrane (colchicine-independent recovery process) and that regeneration of the disrupted distal process of the receptor cell accompanied recovery in the number of available receptor sites (colchicine-dependent recovery process).
Two components of voltage-dependent calcium influx in mouse neuroblastoma cells. Measurement with arsenazo III.
Authors: Bolsover S R
Published: May 2004

N1E-115 mouse neuroblastoma cells were injected with the calcium indicator dye arsenazo III. Optical absorbance changes during voltage-clamp depolarization were used to examine the properties of the two calcium currents present in these cells. The rapidly inactivating calcium current (Moolenar and Spector, 1979b, Journal of Physiology, 292:307-323) inactivates by a voltage-dependent mechanism. The slowly inactivating calcium current is dominant in raising intracellular calcium during depolarizations to greater than -20 mV. Lowering the extracellular calcium concentration affects the two calcium currents unequally, with the slowly inactivating current being reduced more. Intracellular calcium falls very slowly (tau greater than 1 min) after a depolarization. The rapidly inactivating calcium current is responsible for a calcium action potential under physiological conditions. In contrast, it is unlikely that the slowly inactivating calcium current has an important electrical role. Rather, its function may be to add a further increment of calcium influx over and above the calcium influx through the rapidly inactivating calcium channels.
Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells.
Authors: Hess P , Lansman J B , Tsien R W
Published: May 2004

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.
Relationships between the sarcoplasmic reticulum and sarcolemmal calcium transport revealed by rapidly cooling rabbit ventricular muscle.
Authors: Bridge J H
Published: May 2004

Rabbit right ventricular papillary muscles were cooled from 30 to approximately 1 degree C immediately after discontinuing electrical stimulation (0.5 Hz). This produced a contracture that was 30-50% of the preceding twitch magnitude and required 20-30 s to develop. The contractures were identical in cooling solutions with normal (144 mM) or low (2.0 mM) Na. They were therefore not Na-withdrawal contractures. Contracture activation was considerably slower than muscle cooling (approximately 2.5 s to cool below 2 degrees C). Cooling contractures were suppressed by caffeine treatment (10.0 mM). Rapid cooling did not cause sufficient membrane depolarization (16.5 +/- 1.2 mV after 30 s of cooling) to produce either a voltage-dependent activation of contracture or a gated entry of Ca from the extracellular space. Contractures induced by treating resting muscles with 5 X 10(-5) M strophanthidin at 30 degrees C exhibited pronounced tension noise. The Fourier spectrum of this noise revealed a periodic component (2-3 Hz) that disappeared when the muscle was cooled. Cooling contractures decayed with rest (t1/2 = 71.0 +/- 9.3 s). This decay accelerated in the presence of 10.0 mM caffeine and was prevented and to some extent reversed when extracellular Na was reduced to 2.0 mM. 20 min of rest resulted in a net decline in intracellular Ca content of 1.29 +/- 0.38 mmol/kg dry wt. I infer that cooling contractures are principally activated by Ca from the sarcoplasmic reticulum (SR). The properties of these contractures suggest that they may provide a convenient relative index of the availability of SR Ca for contraction. The rest decay of cooling contractures (and hence the decay in the availability of activating Ca) is consistent with the measured loss in analytic Ca during rest. The results suggest that contraction in heart muscle can be regulated by an interaction between sarcolemmal and SR Ca transport.
Guanidine block of single channel currents activated by acetylcholine.
Authors: Dwyer T M
Published: May 2004

The acetylcholine-activated channel of chick myotube was studied using the patch-clamp method. Single channel current amplitudes were measured between -300 and +250 mV in solutions containing the permeant ions Cs+ and guanidine (G+). G+ has a relative permeability, PG/PCs, of 1.6, but carries no more than half the current that Cs+ does, with an equivalent electrochemical driving force. Experiments using G+ revealed an asymmetry of the acetylcholine-activated channel, with G+ being more effective at reducing Cs+ currents when added to the outside than when added to the inside. The block caused by outside, but not inside, G+ was evident for both inward and outward currents. The block caused by outside G+ was voltage dependent, first increasing and then being partially relieved when the driving force was made more negative. Experiments with mixtures of Cs+ and G+ revealed anomalously low magnitudes for reversal potentials, relative to predictions based on the Goldman-Hodgkin-Katz equation. These findings are consistent with a two-well, three-barrier Eyring rate model for ion flow, and demonstrate that a highly permeant ion, guanidine, can block asymmetrically by acting from within the voltage field of the acetylcholine-activated channel.
Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors.
Authors: Mangini N J , Pepperberg D R , Baehr W
Published: May 2004

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.
Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide.
Authors: Maylie J , Irving M , Sizto N L , Boyarsky G , Chandler W K
Published: May 2004

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool solutions. The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1.75 x 10(-6) cm2/s at 18 degrees C. The shape of the resting absorbance spectrum suggests that a fraction 0.11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca. After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign. The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 microM. These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator. Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum. The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution. The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum. A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal. The estimated myoplasmic free [Ca] transient has an average peak value of 26 microM at 18 degrees C. Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143).
Transduction persists in rod photoreceptors after depletion of intracellular calcium.
Authors: Nicol G D , Kaupp U B , Bownds M D
Published: May 2004

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.
Cell suspensions from porcine olfactory mucosa. Changes in membrane potential and membrane fluidity in response to various odorants.
Authors: Kashiwayanagi M , Sai K , Kurihara K
Published: May 2004

A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined. The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium. Various odorants depolarized the cell suspension in a dose-dependent fashion. The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants. The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes [8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)], which suggests that the odorants having different odors are adsorbed on different sites in the membrane. On the basis of these results, a possible mechanism of odor discrimination is discussed.
Single Na+ channels activated by veratridine and batrachotoxin.
Authors: Garber S S , Miller C
Published: May 2004

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.
Sodium channel gating currents. Origin of the rising phase.
Authors: Stimers J R , Bezanilla F , Taylor R E
Published: May 2004

There has been some uncertainty in the past as to the origin of the rising phase of the gating current. We present evidence here that proves that the gating current does not have a rising phase and that the observed rising phase is due to an uncompensated series resistance in the Frankenhaeuser-Hodgkin (F-H) space. When a squid giant axon is bathed in a solution that is 10-20% hyperosmotic with respect to the internal solution, the rising phase of the gating current is eliminated. In parallel with this, a component of the capacity transient (time constant, 20 microseconds) is reduced so that the capacity transient now appears to be closer to a single fast (5-10 microseconds) component. These changes in the capacity transient and gating current occur without altering the amount of charge moved in either. This indicates that the charge is simply redistributed in time. The gating current without a rising phase can still be immobilized by inactivation. Supporting evidence is provided by measuring the accumulation and washout of K+ from the F-H space. It was found that K+ washes out 35% faster when the axon is bathed in hyperosmotic solution. It was estimated that the F-H space thickness (theta) increased 2.5 +/- 0.4-fold (mean +/- SEM) in hyperosmotic solution. Similarly, K+ accumulation in the F-H space was decreased, leading to an estimate of a 5 +/- 1.4-fold increase in theta in hyperosmotic solution. These results are consistent with the simple structural model presented.
Spatiotemporal frequency responses of cat retinal ganglion cells.
Authors: Frishman L J , Freeman A W , Troy J B , Schweitzer-Tong D E , Enroth-Cugell C
Published: May 2004

Spatiotemporal frequency responses were measured at different levels of light adaptation for cat X and Y retinal ganglion cells. Stationary sinusoidal luminance gratings whose contrast was modulated sinusoidally in time or drifting gratings were used as stimuli. Under photopic illumination, when the spatial frequency was held constant at or above its optimum value, an X cell's responsivity was essentially constant as the temporal frequency was changed from 1.5 to 30 Hz. At lower temporal frequencies, responsivity rolled off gradually, and at higher ones it rolled off rapidly. In contrast, when the spatial frequency was held constant at a low value, an X cell's responsivity increased continuously with temporal frequency from a very low value at 0.1 Hz to substantial values at temporal frequencies higher than 30 Hz, from which responsivity rolled off again. Thus, 0 cycles X deg-1 became the optimal spatial frequency above 30 Hz. For Y cells under photopic illumination, the spatiotemporal interaction was even more complex. When the spatial frequency was held constant at or above its optimal value, the temporal frequency range over which responsivity was constant was shorter than that of X cells. At lower spatial frequencies, this range was not appreciably different. As for X cells, 0 cycles X deg-1 was the optimal spatial frequency above 30 Hz. Temporal resolution (defined as the high temporal frequency at which responsivity had fallen to 10 impulses X s-1) for a uniform field was approximately 95 Hz for X cells and approximately 120 Hz for Y cells under photopic illumination. Temporal resolution was lower at lower adaptation levels. The results were interpreted in terms of a Gaussian center-surround model. For X cells, the surround and center strengths were nearly equal at low and moderate temporal frequencies, but the surround strength exceeded the center strength above 30 Hz. Thus, the response to a spatially uniform stimulus at high temporal frequencies was dominated by the surround. In addition, at temporal frequencies above 30 Hz, the center radius increased.
Inhibition of sodium currents by local anesthetics in chloramine-T-treated squid axons. The role of channel activation.
Authors: Wang G K , Brodwick M S , Eaton D C , Strichartz G R
Published: May 2004

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.
Cynomolgus and rhesus monkey visual pigments. Application of Fourier transform smoothing and statistical techniques to the determination of spectral parameters.
Authors: Hárosi F I
Published: May 2004

Microspectrophotometric measurements were performed on 217 photoreceptors from cynomolgus, Macaca fascicularis, and rhesus, M. mulatta, monkeys. The distributions of cell types, for rods and blue, green, and red cones were: 52, 12, 47, and 44, respectively, for the cynomolgus, and 22, 4, 13, and 13 for the rhesus. Visual cells were obtained fresh (unfixed), mounted in various media (some containing 11-cis-retinal), and then located visually under dim red (650 nm) illumination. Absolute absorbance (A), linear dichroism (LD), and bleaching difference (BD) absorbance spectra were recorded through the sides of outer segments. The spectra were subjected to rigorous selection criteria, followed by digital averaging and Fourier transform filtering. Statistical methods were also applied to the accepted samples in the estimation of population means and variances. The wavelength of mean peak absorbance (lambda max) and the standard error at 95% certainty of the rod and blue, green, and red cone pigments in cynomolgus were 499.7 +/- 2.5, 431.4 +/- 4.2, 533.9 +/- 2.4, and 565.9 +/- 2.8 nm, respectively. The rhesus pigments were statistically indistinguishable from the cynomolgus, having lambda max of approximately 500, 431, 534, and 566 nm. Statistical tests did not reveal the presence of a lambda max subpopulation (i.e., anomalous pigments). The bandwidth of each alpha-band was determined in two segments, giving rise to the longwave half-density (LWHDBW), shortwave half-density (SWHDBW), and total half-density (THDBW) bandwidths. The LWHDBW was found to have the smallest variance. Both the LWHDBW and the THDBW showed linear dependence on the peak wavenumber (lambda max)-1 for the four macaque pigments.
The response to monochromatic light flashes of the oxygen consumption of honeybee drone photoreceptors.
Authors: Jones G J , Tsacopoulos M
Published: May 2004

Local measurements of the fall in oxygen pressure on stimulation of slices of the retina of the honeybee drone by flashes of light were made with oxygen microelectrodes and used to calculate the kinetics of the extra oxygen consumption (delta QO2) induced by each flash. The action spectrum for delta QO2 was obtained from response-intensity curves in response to brief (40 ms) monochromatic light flashes. The action spectrum of receptor potentials was obtained with the same experimental conditions. The two action spectra match closely: they deviate slightly from the photosensitivity spectrum of the drone rhodopsin (R). The deviation is thought to be due to wavelength-dependent light scattering and absorption in the preparation. In these experiments, the visual pigment was first illuminated with orange light, which is known to convert the bistable drone photopigment predominantly to the R state from the metarhodopsin (M) state. When long (300-900 ms) light flashes were used to elicit delta QO2, the responses to different wavelengths could not be matched in time course (as for the short flashes). Flashes producing large R-to-M conversions produced a prolonged delta QO2. The prolongation did not occur after double flashes, which produced both large R-to-M and M-to-R conversions. Similar changes in the length of afterpotentials in the photoreceptor cells and in a long-lasting decrease in photoreceptor intracellular K+ activity were found after long single or double flashes. The results are interpreted to show that the initial event for stimulation by light of metabolism in the drone retina is the same as that for stimulation of electrical responses (i.e., absorption of photons by R). Absorption of photons by M can produce an inhibitory effect on this stimulation.
Patch-clamp studies of isolated mouse olfactory receptor neurons.
Authors: Maue R A , Dionne V E
Published: May 2004

Olfactory receptor neurons isolated from embryonic, neonatal, and adult mice were studied using the patch-clamp technique. Several distinct types of ion channels were characterized in patches of membrane from the neuronal soma and the dendritic knob of receptor neurons, including a 130-pS Ca++-activated K+ channel with voltage-dependent kinetics, an 80-pS Ca++-activated K+ channel with voltage-insensitive kinetics, a 25-pS K+ channel with properties similar to inward rectifiers, and a 40-pS K+ channel that was activated and then inactivated by rapid depolarization. Evidence of large-conductance (greater than 200 pS) Cl- channels, which were Ca++ insensitive and increasingly active at depolarizing membrane potentials, and voltage-activated Ca++ channels (16 pS) was also obtained. From K+ channel activity recorded from cell-attached patches, the intracellular [Ca++] was inferred to be below 0.1 microM, and the membrane potential was inferred to be approximately -50 mV. The receptor neurons had high input resistances, and action potentials could be elicited by picoampere amounts of depolarizing current. The receptor neurons responded to applied odorant molecules and to forskolin with increases in membrane conductance. These results provide a description of the membrane properties of olfactory receptor neurons and a basis for understanding their electrical activity and response to odorants.
Dynamics of the ganglion cell response in the catfish and frog retinas.
Authors: Sakuranaga M , Ando Y , Naka K
Published: May 2004

Responses were evoked from ganglion cells in catfish and frog retinas by a Gaussian modulation of the mean luminance. An algorithm was devised to decompose intracellularly recorded responses into the slow and spike components and to extract the time of occurrence of a spike discharge. The dynamics of both signals were analyzed in terms of a series of first-through third-order kernels obtained by cross-correlating the slow (analog) or spike (discrete or point process) signals against the white-noise input. We found that, in the catfish, (a) the slow signals were composed mostly of postsynaptic potentials, (b) their linear components reflected the dynamics found in bipolar cells or in the linear response component of type-N (sustained) amacrine cells, and (c) their nonlinear components were similar to those found in either type-N or type-C (transient) amacrine cells. A comparison of the dynamics of slow and spike signals showed that the characteristic linear and nonlinear dynamics of slow signals were encoded into a spike train, which could be recovered through the cross-correlation between the white-noise input and the spike (point process signals. In addition, well-defined spike correlates could predict the observed slow potentials. In the spike discharges from frog ganglion cells, the linear (or first-order) kernels were all inhibitory, whereas the second-order kernels had characteristics of on-off transient excitation. The transient and sustained amacrine cells similar to those found in catfish retina were the sources of the nonlinear excitation. We conclude that bipolar cells and possibly the linear part of the type-N cell response are the source of linear, either excitatory or inhibitory, components of the ganglion cell responses, whereas amacrine cells are the source of the cells' static nonlinearity.
Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons.
Authors: Shuster M J , Siegelbaum S A
Published: May 2004

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.
Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents.
Authors: Niles W D , Cohen F S
Published: May 2004

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.
Gating kinetics of four classes of voltage-dependent K+ channels in pheochromocytoma cells.
Authors: Hoshi T , Aldrich R W
Published: May 2004

Clonal pheochromocytoma (PC-12) cells have four different types of voltage-dependent K+ channels whose activation does not require high concentrations of Ca++ on the cytoplasmic side of the membrane (Hoshi, T., and R. W. Aldrich, 1988, Journal of General Physiology, 91:73-106). The durations of open and closed events of these four different types of voltage-dependent K+ channels were measured using the excised configuration of the patch-clamp method. The open durations of a class of K+ channels termed the Kz channel, which activates rapidly and inactivates slowly in response to depolarizing pulses, had two exponential components. The closed durations of the Kz channel had at least four exponential components. The time constants of the fastest of the two exponential components in the closed durations were very similar to those of the two exponential components present in the first-latency distribution. The first latencies of the Kz channel decreased steeply with depolarization, contributing to the increased probability of the channel being open with depolarization. The Kz channel also had a very slow gating process that resulted in a clustering of blank sweeps. A gating scheme containing two open states and five closed states is consistent with the observations. The Ky channel had one exponential component in the open durations and three exponential components in the closed durations. The first latencies varied greatly depending on the prepulse voltage and duration. The results were consistent with a sequential model with a large number of closed states and one open state. The Kx channel, which required large hyperpolarizing prepulses to remove steady state inactivation and did not show inactivation with maintained depolarization, had two exponential components in the open durations and three exponential components in the closed durations. The burst behavior of the Kx channel involved many more than two states. The transient Kw channel had one exponential component in the open durations and the mean open time increased with depolarization. The first latencies of the Kw channel were steeply dependent on the voltage, decreasing with depolarization.
Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.
Authors: Drapeau P , Nachshen D A
Published: May 2004

We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.
Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase.
Authors: Brozovich F V , Yates L D , Gordon A M
Published: May 2004

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.
Veratridine modifies open sodium channels.
Authors: Barnes S , Hille B
Published: May 2004

The state dependence of Na channel modification by the alkaloid neurotoxin veratridine was investigated with single-channel and whole-cell voltage-clamp recording in neuroblastoma cells. Several tests of whole-cell Na current behavior in the presence of veratridine supported the hypothesis that Na channels must be open in order to undergo modification by the neurotoxin. Modification was use dependent and required depolarizing pulses, the voltage dependence of production of modified channels was similar to that of normal current activation, and prepulses that caused inactivation of normal current had a parallel effect on the generation of modified current. This hypothesis was then examined directly at the single-channel level. Modified channel openings were easily distinguished from normal openings by their smaller current amplitude and longer burst times. The modification event was often seen as a sudden, dramatic reduction of current through an open Na channel and produced a somewhat flickery channel event having a mean lifetime of 1.6 s at an estimated absolute membrane potential of -45 mV (23 degrees C). The modified channel had a slope conductance of 4 pS, which was 20-25% the size of the slope conductance of normal channels with the 300 mM NaCl pipette solution used. Most modified channel openings were initiated by depolarizing pulses, began within the first 10 ms of the depolarizing step, and were closely associated with the prior opening of single normal Na channels, which supports the hypothesis that modification occurs from the normal open state.
Light-induced oxygen consumption in Limulus ventral photoreceptors does not result from a rise in the intracellular sodium concentration.
Authors: Fein A , Tsacopoulos M
Published: May 2004

Illumination of Limulus ventral photoreceptors leads to an increase in the intracellular concentration of sodium, [Na+]i, and to an increase in the consumption of O2 (delta QO2). After a 1-s light flash, it takes approximately 480 s for [Na+]i to return to within 10% of its preillumination level, whereas delta QO2 takes approximately 90 s. Thus, the delta QO2 is complete long before [Na+]i has returned to its resting level. Pressure injection of Na+ into the cell in order to elevate [Na+]i to the same levels as attained by illumination causes a rise in [Na+]i that returns to baseline with the same time course as the light-induced rise in [Na+]i. However, the injection of Na+ does not lead to an increase of the consumption of O2. We conclude that activation of the Na pump by a rise in [Na+]i is not a factor involved in the light-induced activation of O2 consumption in these cells.