Voltage Dependence of the Glycine Receptor–Channel Kinetics in the Zebrafish Hindbrain

Voltage Dependence of the Glycine Receptor–Channel Kinetics in the Zebrafish Hindbrain PASCAL LEGENDRE Institut des Neurosciences, Université Pierre et Marie Curie, 75252 Paris Cedex 05, France Legendre, Pascal. Voltage dependence of the glycine receptor– channel kinetics in the zebrafish hindbrain. J. Neurophysiol. 82: 2120 –2129, 1999. Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outsideout current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from 250 to 120 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of '5 and '30 ms (holding potential of 250 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 6 2.8% of the total current at 250 mV and 54.1 6 10% at 120 mV. The 20 – 80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20 – 80% rise time was 0.2 ms at 270 mV and 0.22 ms at 120 mV. Responses evoked by 100 –200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested. INTRODUCTION Multiple voltage-dependent postsynaptic mechanisms modulate the activity of ligand-gated channels responsible for excitatory and inhibitory postsynaptic currents. Such mechanisms may function to control the postsynaptic efficacy of synaptic events (Faber and Korn 1987) or may operate to prevent cell damage due to excessive depolarization, as for excitatory glutamatergic synaptic events (Collingridge and Lester 1989; Rothman and Choi 1990). In most cases, voltage dependence results from open channel blockade by ions such as Mg21 for N-methyl-D-aspartate (NMDA) receptors (Mayer et al. 1984; Nowak et al. 1984) or by the neurotransmitter itself, as shown The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2120 for the nicotinic acetylcholine receptor (Liu and Dilger 1991; Ogden and Colquhoun 1985; Sine et al. 1990, Sine and Steinbach 1984). It can also be due to voltage-dependent changes in ion permeation, as for some kainate receptor subtypes (Curutchet et al. 1992), central nicotinic receptors (Mulle and Changeux 1990) or 5-HT3 receptors (Derkach et al. 1989). But this can also be related to intrinsic voltage-dependent gating behavior of the receptor channel itself as, for example, the voltage-dependent desensitization described for GABAA and glycine receptors (Akaike and Kaneda 1989; Bormann et al. 1987; Burgard et al. 1996, Dominguez-Perrot et al. 1996; Gunderson et al. 1984, 1986; Mellor and Randall 1998). Voltage dependence of glycinergic inhibitory postsynaptic currents (IPSCs) duration was first described in the Mauthner cell (M-cell) of the goldfish (Faber and Korn 1987). A similar property of glycine responses was reported in larva and adult zebrafish M-cell (Hatta and Korn 1998; Legendre and Korn 1995) and in mammalian neurons in slices (Otis and Mody 1992; Stuart and Redman 1990). However, the GlyRs gating properties involved have not yet been elucidated. In the zebrafish hindbrain an increase in miniature IPSC (mIPSC) duration with membrane depolarization is correlated with the increase in GlyRs opening burst duration (Legendre and Korn 1995), suggesting that fast GlyRs kinetics can be voltage sensitive. Recent analysis of the gating behavior of GlyRs using fast-flow application techniques on outside-out patches had revealed a complex behavior of the zebrafish glycine-operated channels (Legendre 1998). In the zebrafish hindbrain, the decay time of mIPSCs is controlled by gating modes (a reluctant and a willing gating mode) closely similar to those described for the bullfrog N-type calcium channel (Bean 1989; Boland and Bean 1993; Elmslie et al. 1990; Elmslie and Jones 1994). The interconversion between these two gating modes is voltage dependent for the bullfrog N-type calcium channel (Boland and Bean 1993). This might also be the case for GlyRs as the GlyRs Markov model predicts that an increase of the rate constant from the doubly liganded closed state to the reluctant closed state can greatly enhance the duration of mIPSCs (Legendre 1998). However, changes in the time course of the deactivation phase of a mIPSC might also result from a change in the closing rate constant and/or the dissociation rate constant (Legendre 1998). To address this issue I took advantage of the M-cell of the 50-h-old zebrafish (Danio rerio) brain preparation (Legendre and Korn 1994). I analyzed the voltage-dependent gating properties of the glycine receptors (GlyR) using fast-flow application techniques (Franke et al. 1987; Lester et al. 1990) and 0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. VOLTAGE-DEPENDENT KINETICS OF THE GLYCINE RECEPTOR outside-out recordings to unravel the voltage-dependent channel gating reactions. By comparing my experimental data to simulated traces obtained from GlyRs Markov model, I demonstrate that changes in glycinergic mIPSCs duration with membrane potential are likely to result from a voltage dependence of closing rate constants for the GlyR channel. The origins of the voltage dependance of closing rate constants are discussed with respect to anion permeation versus charged moieties in receptor subunits that can move with respect to the electrical field when the channel gates. 2121 coefficient for glycine close to 0.5–1 3 1025 cm2s21, the estimated absolute exchange time was found to be #0.1 ms. Outside-out patch current analysis The isolated intact zebrafish brain was prepared as previously described (Legendre and Korn 1994). Briefly, the brains of 50-h-old larvae were dissected out and glued to a coverslip using a plasmathrombin embedding procedure. Before starting the experiments, brain preparations were stored for 15 min in an oxygenated (95% O2-5% CO2) bathing solution containing (in mM) 145 NaCl, 1.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose, with the osmolarity adjusted to 330 mOsm. Single-channel currents were filtered at 10 kHz using an eight-pole Bessel filter (Frequency Devices), sampled at 50 kHz (Digidata 1200 interface, Axon Instruments), stored on an IBM AT compatible computer using Pclamp software 6.03 (Axon Instruments) and analyzed off-line with Axograph 3.5 software (Axon Instruments). The time courses of outside-out responses was analyzed by averaging 10 –15 single events using Axograph 3.5 (Axon Instruments; filter cutoff frequency: 10 kHz). The activation time constants of currents evoked by a low concentration of glycine 0.1– 0.03 mM glycine applications (100 –200 ms) were estimated by fitting the onset of the responses with a sum of two sigmoidal curves (Legendre 1998) using Axograph 4 software (filter cutoff frequency: 10 kHz). To fit the rise time of these responses, their onset was determined from that of the chloride currents evoked by the application of a saturating concentration (3–10 mM) of glycine (Legendre 1998). The first 150 ms of the decay phase of the outside-out currents evoked by a brief (1 ms) application of 3–10 mM glycine was fitted with a sum of two exponential curves to determine their decay time constants (Legendre 1998). Outside-out patch-clamp recordings Kinetic modeling programs Standard outside-out recordings (Hamill et al. 1981) were achieved under direct visualization (Nikon Optiphot microscope) on the M-cell located in the fourth hindbrain rhombomere (Metcalfe et al. 1986) as previously described (Legendre 1998). The isolated brain was continuously perfused at room temperature (20°C) with the oxygenated bathing solution (2 ml/min) in the recording chamber (0.5 ml). Patchclamp electrodes were pulled from thick-wall (10 –15 MV) borosilicate glass. They were fire-polished and filled with (in mM) 135 CsCl, 2 MgCl2, 4 Na3ATP, 10 EGTA, 10 HEPES, pH 7.2. The osmolarity was adjusted to 290 mOsm. Outside-out patches were obtained by slowly pulling the pipettes out of the brain. The resistance of outsideout patches ranged from 2 to 10 GV. Currents were recorded using an Axopatch 1D amplifier (Axon instruments), filtered at 10 kHz, and stored using a digital tape recorder (DAT DTR 1201, SONY). The kinetic model for GlyR behavior we used was previously determined for M-cell GlyRs (Legendre 1998). Glycine-evoked currents were analyzed off-line using chemical kinetic modeling programs (Axograph 4, Axon Instruments) on a Power Macintosh (7600/ 132) to adjust the rate constants to obtain theoretical responses with time course similar to the experimental data. This program first calculated the evolution of the number of channels in each given state for given rate constants. Simulated traces were obtained using Axogaph 4 software by varying one rate constant with voltage according to the experimental measurements. Patch currents represent the average of $10 traces as specified in the figure legends or the text. Results are presented as means 6 SD throughout unless otherwise noted. Drug delivery I examined the voltage dependence of the activation and deactivation kinetics of native glycine receptors obtained from the Mauthner cell (M-cell) using fastflow application techniques. Two types of glycinergic receptors have been functionally characterized on the zebrafish M-cell (Legendre 1997). They represent the expression of homomeric-like a1 and heteromeric-like a/b receptors (Legendre 1997). These two receptors can be discriminated by their mean conductance states and the number of their subconductance levels. In the present study, I focused my analysis on heteromeric-like receptors characterized by a single conductance state of 40 – 46 pS because their general kinetic properties have been previously determined (Legendre 1998). Patches containing channels with a main conductance state of 80 – 86 pS and multiple subconductance levels were therefore omitted. METHODS Isolated intact brain preparation Outside-out single-channel currents were evoked using a fast-flow application system (Franke et al. 1987; Legendre 1998; Lester et al. 1990). Drugs were dissolved in a control solution containing (in mM) 145 NaCl, 1.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.2, osmolarity 330 mOsm. Control and drug solutions were gravity fed into the two channels of a thin-wall glass theta tube (2 mm OD, Hilgenberg, Germany) pulled and broken to obtain a tip diameter of 200 mm. One lumen of the tube was connected to reservoirs filled with solutions containing different glycine concentrations. The solution exchange was performed by rapidly moving the solution interface across the tip of the patch pipette, using a piezoelectric translator (Physics Instrument, model P245.30). Concentration steps of glycine lasting 1–200 ms were applied every 5–10 s. The exchange time (0.08 ms) was determined after rupturing the seal by monitoring the change in the liquid junction evoked by the application of a control solution diluted by 10% to the open tip of the patch pipette (Legendre 1998). As the absolute exchange on the patch partially results from an unstirred layer around the patch, the theoretical limit to the speed of solution change was estimated using the method published by Maconochie and Knight (1989) (see Legendre 1998, for detailed analysis). Assuming that the patch has a spherical geometry with a diameter of 0.5 mm (patch electrode resistance .10 MV) and a diffusion RESULTS Time course of 3 mM evoked outside-out currents with voltage Transient outside-out currents evoked by a short step into a saturating concentration of glycine have closely similar time course to that of mIPSCs recorded in the zebrafish M-cell Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. 2122 P. LEGENDRE (Legendre 1998). The deactivation phase of these currents could be fitted by the sum of two exponential curves with decay time constants of '5 ms and '30 ms (Vh 5 250 mV). The double exponential deactivation results from the complex gating behavior of GlyRs (Legendre 1998) (see Fig. 5A). Several mechanisms might underlie the voltage-dependent increase in mIPSC decay time and the single-channel opening burst durations (Legendre and Korn 1995). It can result from a decrease in the closing rate constant, interconversion between gating modes, or a decrease in the dissociation rate constant. Analysis of the time course of glycine-evoked responses with voltage would thus give information about the possible voltage-dependent gating reactions (Legendre 1998). The basis of the voltage dependence of glycine-gated channels was first investigated by the analysis of the activation and deactivation kinetics of the patch currents evoked by a short application step (1 ms) into 3 mM of glycine. Increasing the holding potential (Vh) from 260 to 120 mV did not change significantly the maximum chloride conductance measured at the peak of the responses evoked by 1-ms applications of 3 mM glycine. A linear current-voltage relationship was obtained in all patches tested (n 5 10) when Vh was increased from 250 to 120 mV (Fig. 1). For Vh less than 250 mV, a small deviation of the recorded current amplitude from the linear regression line was observed (Fig. 1B). This slight decrease in the maximum macroscopic conductance with low voltages is likely to be due to rectification of GlyRs microscopic conductance, as previously described on patches pulled from the zebrafish M-cell (Legendre and Korn 1994). The deactivation phase of outside-out currents evoked by a saturating concentration of glycine is voltage dependent. When Vh was increased, the deactivation phase of these responses was prolonged (Fig. 2A). It remained biphasic at all membrane potentials tested and could be well fitted by the sum of two exponential curves (Figs. 2 and 3). Short and long decay time constants were tfast 5 5.1 6 0.53 ms and tslow 5 41.4 6 7.8 ms (n 5 10) at Vh 5 250 mV. When the patches were depolarized to 120 mV, tfast and tslow significantly increased to reach 9.8 6 0.97 ms and 67.7 6 11.5 ms (mean 6 SD, n 5 8), respectively (paired t-test, P 5 0.01). tfast increased progressively with voltage (Fig. 2B). The relationship between tfast and the holding potential can be fitted by a single exponential function between 250 and 120 mV given an increase in tfast with a limiting slope of e-fold/95 mV (Fig. 2C). tslow also increased progressively with voltage (Fig. 3A). Assuming that tslow also changed exponentially when membrane potential was increased, we found that tslow increased e-fold per 111 mV/between 250 and 10 mV. This is closely similar to that obtained for tfast. In contrast to the decay time constants, the relative amplitude of these two decay components was not significantly voltage dependent (paired t-test, P 5 0.1). At Vh 5 250 mV, the fast decay component represented 64.2 6 2.8% (n 5 10) of the total current while depolarizing the patch to 120 mV slightly decreased its relative amplitude to 54.1 6 10% (n 5 8; Fig. 3B). The voltage sensitivity of the two decay time constants with voltage and the lack of voltage dependence of their relative proportion might result from voltage-dependent opening rate constants. To test this hypothesis, we analyzed the activation phase of the transient current evoked by 1-ms application of a FIG. 1. Current-voltage relationship of current transients evoked by a short (1 ms) application of a saturating concentration of glycine. A: superimposed traces of 3 mM glycine evoked responses recorded from an outside-out patch obtained at different holding potentials (Vh). Each trace represents the average of 10 epochs (filter cutoff frequency 5 4 kHz). B: current-voltage curve obtained from data shown in A. Measurement of the peak current was performed on averaged traces. Note that current-voltage relationship is linear when Vh was depolarized from 250 to 120 mV. saturating concentration of glycine (3 mM). When a saturating concentration of agonist is applied, the limiting factor for the rise time of the evoked currents becomes the opening rate and the closing rate constants (b1 and a1 for GlyRs, respectively) linking the open state and the doubly liganded closed state. According to the GlyR Markov model (Legendre 1998) shown in Fig. 5A, changes in the rise time constant ton [ton 51/(b1 1 a1)] with voltage can give information on the voltage sensitivity of these rate constants. But, as the opening rate constant b1 of GlyRs is .10 times faster than the closing rate constant a1 (b1 ' 9,000 s21; a1 ' 600 –700 s21) (Legendre 1998), any modifications in the onset duration will mainly reflect fluctuations in the opening rate constant b1. For example a two time change in a1 will modify ton by '4% only. To estimate the voltage dependence of the opening rate constant b1, I measured the 20 – 80% rise time of the responses evoked by 1-ms application of 3 mM glycine. As shown in Fig. 3, C and D, the 20 – 80% rise time did not change with voltage. The 20 – 80% rise time measured at Vh 5 270 mV (0.2 6 0.012 ms; n 5 6) or Vh 5 250 mV (0.19 6 0.01 ms; n 5 6) was not significantly modified (paired t-test, P 5 0.1) when the Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. VOLTAGE-DEPENDENT KINETICS OF THE GLYCINE RECEPTOR 2123 be due to voltage sensitivity of the dissociation rate constant koff. As these rate constants also control the rising phase of responses evoked by a nonsaturating concentration of the agonist (Legendre 1998), I analyzed the activation phase of the outside-out currents evoked by 0.1 mM glycine applications at membrane potentials ranging from 260 mV to 120 mV. The activation phase of 0.1 mM glycine-evoked responses has a sigmoidal onset corresponding to the presence of two binding sites. The biphasic time course reflects equilibration between the two opening gating modes of GlyRs (Legendre 1998). The activation phases of these responses was better fitted with a sum of two sigmoidal functions of the form $ap@1 2 exp~2t/ t on1 !# 2 % 1 $bp@1 2 exp~2t/ t on2 !# 2 % where a and b are the relative amplitudes of the two components and ton1 and ton2 are the corresponding time constants (Fig. 4A) (Legendre 1998). Activation time constants were analyzed from responses evoked by 100- to 200-ms pulse applications of 0.1 mM glycine to ensure glycine-binding equilibrium at the peak of the responses. Increasing the holding potential from 260 to 20 mV did not significantly change the fast (ton1) and the slow (ton2) time constants of the two components of the activation phase (paired t-test, P 5 0.1). ton2 showed a small tendency to increase when Vh was increased (Fig. 4C). When the outside-out patches were held at 250 mV, ton1 and ton2 had a value of 2.45 6 0.31 ms and 8.1 6 1.99 ms (n 5 5), respectively. Changing Vh from 250 to 20 mV gave ton1 and ton2 values of 2.18 6 0.5 ms and 10.5 6 2.32 ms (n 5 5), respectively. The relative amplitude of these two components was concentration dependent (Legendre 1998) but did not change significantly with voltage (paired t-test, P 5 0.1; Fig. 4D). For example, ton1 had a relative amplitude of 0.619 6 0.13 at Vh 5 250 mV and 0.624 6 0.15 at Vh 5 120 mV (n 5 5). These observations imply that transitions between gating reactions linking the willing and reluctant states of the GlyR are not voltage sensitive. They also suggest that the dissociation rate constant koff shows relatively little voltage dependence. Closing rate constants are likely to be voltage dependent FIG. 2. Decay time of outside-out currents evoked by 1-ms application of 3 mM glycine are voltage dependent. A: averaged glycine-evoked traces obtained at Vh 5 250 mV and 120 mV. Deactivation phases can be well described by the sum of 2 exponential curves at both voltages. Note that the response had a longer decay phase at positive voltage. B: plot of the fast decay component (tfast) values vs. holding potential. Each point represents the average of 4 –10 measurements (6SD). Note that tfast increased with Vh. C: semi-logarithmic plot of data shown in B. Limiting voltage sensitivity of tfast (e-fold/95 mV) was measured by fitting the ascending part of the curve between 260 and 210 mV with tfast in Ln scale. outside-out patches were depolarized to 20 mV (0.22 6 0.014 ms; n 5 8). It is therefore unlikely that the opening rate constant b can be voltage dependent. Responses evoked by a nonsaturating concentration of glycine Changes in the deactivation time constants with voltage can also reflect voltage-dependent interconversion between the two GlyR gating modes (i.e., the rate constants linking the willing state A2C and the reluctant state A2C* of Fig. 5A). It can also My experimental data therefore suggests that the closing rate constants a1 and a2 (Fig. 5A) are most likely to be voltage sensitive. But the mean open times cannot be directly estimated from classical stationary analysis of the glycine-gated channel activity due to unresolved short closures (,0.1 ms), which correspond to the fast opening rate constants of GlyRs (Legendre 1998). To determine the voltage sensitivity of these closing rate constants, experimental data, described herein, were therefore compared with simulated outside-out currents using the Markov model previously proposed for zebrafish GlyRs (Legendre 1998) (Fig. 5A). The rate constants were adjusted to construct simulated traces with time courses similar to experimental measurements performed at Vh 5 250 mV (see Fig. 5). Two different kinetic models were tested: in one closing rate constants and in the other dissociation rate constants were given a voltage dependence. This comparison was done because the two times change in the decay time constants of outside-out current between 250 and 120 mV might also result from a slight voltage sensitivity of the dissociation rate Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. 2124 P. LEGENDRE FIG. 3. A: 2nd decay component (tslow) also appeared to be voltage dependent. Each point represents the average of 4 –10 measurements (6SD). B: relative amplitude of the 2 components of the deactivation phase (tfast and tslow) at different holding potentials. Note that the relative amplitude of tfast and tslow is not strongly voltage-dependent. Each point represents the average of 4 –10 measurements (6SD). C: superimposed activation phase of normalized responses evoked by the application of 3 mM glycine obtained at Vh 5 250 mV and Vh 5 120 mV. Note that these 2 activation phases are not different. D: 20 – 80% rise time vs. holding potential obtained from responses evoked by 1 ms duration application of 3 mM glycine. Each point represents the average of 4 –10 experiments. Note that the 20 – 80% rise time is not voltage dependent (filter cutoff frequency: 10 kHz). constant koff as predicted by the GlyR Markov model shown in Fig. 5A (Legendre 1998). Moreover, this Markov model predicts that a two times change in the koff value will have a limited effect on the activation time course of responses evoked by the application of a low concentration of glycine (Legendre 1998). The complex deactivation phase of transient currents evoked by a short pulse of glycine corresponds to clusters of bursts of channel openings arising from the two open states O1 and O2 linked to the willing state A2C and the reluctant state A2C*, respectively (Legendre 1998). The number of openings per burst arising from O1 can be influenced by changes in the koff value with respect to the opening rate constant b and the rate constant d between A2C and A2C* [N 5 1 1 (b1/d 1 koff)]. Decreasing koff will also decrease the probability of escape from the reluctant gating mode, which will, in turn, increase the duration of the clusters of bursts of openings arising from O2 while burst duration remains unchanged. This results because the number of openings per burst (N) arising from O2 is not modified because it depends primarily on the opening rate constant b2 and the reverse rate constant r between A2C* and A2C [N 5 1 1 (b2/r)]. Finally, a model in which the opening rate constants b were given a voltage dependence is unlikely. Such a model predicts a '40% decreased in the 20 – 80% rise time of responses evoked by 3-mM glycine applications when the patch is depolarized from 250 mV to 120 mV. This was not experimentally observed (Fig. 3, C and D). The first model had two voltage-dependent closing rate constants (a1 and a2) with similar voltage sensitivities, as the two deactivation components tfast and tslow were increased to the same extent by depolarizing the patch to 120 mV (Figs. 2 and 3). Changes in the rate constants a with voltage were calculated using the relation of the form a 5 a 250mV p exp@~2V h 2 50 mV!/95 mV# where a250 mV is the closing rate constant estimated at 250 mV (a1 5 620 s21 and a2 5 1,300 s21). The second model supposes that the dissociation rate constant koff is voltage dependent. Changes in the dissociation rate constant koff with voltage were calculated using the following equation k off 5 k off250 mV p exp@~2V h 2 50 mV!/95 mV# where koff 250 mV is the dissociation rate constant estimated at 250 mV (koff 5 1,550 s21). We first compared the time course of responses to 1-ms application of 3 mM glycine with data from simulations based on these theoretical models. Theoretical and experimental data were compared at Vh from 260 to 120 mV. As shown in Fig. 5B, changes in koff with voltage (model 2) cannot properly describe the voltage-dependent increase of the fast decay component when the membrane is depolarized. In contrast a good agreement between experimental and simulated data were obtained when a was made to be voltage dependent. Changes in a with voltage can also account for the increase of the slow decay component with membrane depolarization (Fig. 5C). Furthermore this model predicts that the relative amplitudes of the two decay components has a little voltage dependence (Fig. 5D). This is not the case when koff was made to be voltage sensitive. An increase in koff with voltage will evoke a decrease in the relative proportion of the fast decay component, which is not the case in my experimental conditions (Fig. 5D). To obtain a similar increase in the decay time constant of the fast deactivation component with Vh depolarized to 120 mV, koff must decrease e-fold/32 mV. In this case, the model predicts that the time constant of the second decay component will increase by four times (160 ms) at Vh 5 120 mV, whereas the relative proportion of the fast decay component will decrease to 23%. This was not experimentally observed. The accuracy of model 1 was confirmed when experimental responses evoked by a low concentration of glycine were Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. VOLTAGE-DEPENDENT KINETICS OF THE GLYCINE RECEPTOR 2125 voltage, as observed experimentally (Fig. 6C). However, model 2 (koff being voltage sensitive) predicts a decrease in the slow time constant of the activation phase component when Vh was increased, which is not the case in my experimental conditions (Fig. 6B). Altogether these results suggest that the change in glycine-evoked transient outside-out current duration with membrane potential may be explained by voltage-dependent closing rate constants a1 and a2, the other gating reactions being voltage insensitive. DISCUSSION The present study shows that the voltage dependence of the duration of glycinergic evoked transient outside-out current can result from an increase in the mean open times of GlyRs. This is consistent with my previous work showing that the deactivation time course of glycinergic mIPSCs depends primarily on the glycine-gated channel kinetics (Legendre 1998). It seems unlikely that this property reflects the presence of postsynaptic immature GlyRs because voltage-dependent IPSCs duration can be observed in the M-cell of the adult zebrafish (Hatta and Korn 1998) and of the adult goldfish (Faber and Korn 1987). Voltage dependence of glycine-evoked outside-out currents FIG. 4. Voltage dependence of the activation time course of outside-out responses evoked by application of 0.1 mM glycine. A: example of normalized averaged trace currents (n 5 10 per trace) evoked by step applications of glycine at Vh 5 250 mV and Vh 5 120 mV. Each 25th data point only is plotted for clarity. The onset of these responses were fitted by the sum of 2 sigmoidal curves given 2 activation time constants (see RESULTS). B: changes in the activation time constants with voltage. Each point represents the average of 5 experiments. Note that the 2 activation phase components were not strongly voltage dependent. C: relationship between the relative amplitude of the 2 sigmoidal components of the activation phases and Vh (each point is the average of 5 measurements). Note that the relative amplitude of these 2 components is insensitive to voltage. compared with theoretical traces obtained from models 1 and 2. As previously mentioned, setting a or koff as voltage-dependent rate constants did not strongly modify the fast activation time constant of responses evoked by the application of 0.1 mM glycine (Fig. 6B). When Vh was depolarized from 260 to 120 mV, the two models predict that the relative proportion of the two activation phase components will be little affected by The peak current evoked by a brief application of a saturating concentration of glycine varied linearly between 260 and 120 mV as do evoked glycinergic synaptic current recorded in the adult goldfish M-cell (Faber and Korn 1987) and in spinal motoneurons of the cat (Stuart and Redman 1990). This is consistent with the lack of voltage sensitivity of the opening rate constants of GlyRs and the maximum open probability (0.9) of glycine-gated channels measured at Vh 5 250 mV (Legendre 1998). The fast decay time constant increased e-fold per 95 mV. This is closely similar to that reported for evoked IPSCs in the cat motoneurons (e-fold/91) (Stuart and Redman 1990), which suggests that hindbrain zebrafish GlyRs share some functional characteristics with mammalian spinal cord receptors. The voltage dependence of the decay time constants is, however, two to three times less than that for glycine-gated channel activity (e-fold/35 mV) recorded under stationary conditions (Legendre and Korn 1995). This is likely to be due to two independent mechanisms (Legendre and Korn 1995), one involving voltage-dependent closing rate constant and the other one reflecting voltage-dependent slow desensitization (Akaike and Kaneda 1989). These observations also imply that desensitization should show a stronger voltage dependence than the opening rate constant. Voltage-dependent desensitization cannot, however, account for the change in duration of the postsynaptic responses with voltage. It develops too slowly, and it cannot shape the time course of mIPSCs or transient outside-out currents evoked by a short application (,50 ms) of glycine (Legendre 1998). A voltage dependence of IPSC duration mediated by changes in channels kinetics is also observed at GABAergic synapses. The deactivation phase of GABA-evoked transient current and the amount of GABAA desensitization are also voltage dependent (Mellor and Randall 1998; Yoon 1994). Although it appears that the increase in the proportion of the Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. 2126 P. LEGENDRE FIG. 5. A: Markov model reproducing the properties of the glycine-gated channel of the zebrafish hindbrain reticular neurons (Legendre 1998). This model possesses 2 equivalent agonist binding steps and the doubly liganded closed state (A2C) provides access to a reluctant closed state A2C*. These 2 doubly liganded closed states can also provide access to 2 independent open states. Traces in A show examples of theoretical responses generated by this model at 2 different voltages, when the 2 closing rates a1 and a 2 have a voltage sensitivity of e-fold/95 mV (see RESULTS). The rate parameters used to generates glycineevoked responses were kon 5 5 mM21s21, koff 5 1,550 s21, a1 5 620 s21, b1 5 8,938 s21, a2 5 1,300 s21, b2 5 3,180 s21, d 5 1,150 s21, and r 5 120 s21). B: analysis of predicted changes in the fast deactivation with voltage when the closing rate (●) or the dissociation rate constant (E) were set to be voltage dependent. The continuous lines represent experimental data shown in Fig. 2B. C: predicted change in the slow deactivation time constant with voltage. As in B models with voltage-dependent closing rate constants (●) or dissociation rate constants (E) were compared with experimental data. D: theoretical changes with voltage in the relative proportion of the 2 components of the deactivation phase, depending on the kinetics model used as explained above. Note that in all cases, only the model with voltagedependent closing rate constants (a1 and a 2) can predict my experimental data. fast desensitized GABA-evoked currents and the increase in GABA-evoked response duration are, as for GlyRs, independent (Mellor and Randall 1998), it seems likely that distinct mechanisms operate at these two receptors. The biphasic deactivation of GABAergic responses is controlled by a fast desensitization mechanism (Jones and Westbrook 1995) that increases at depolarized potentials in cerebellar granule cells (Mellor and Randall 1998). To the contrary, the amount of desensitized current evoked by glycine application is decreased when the membrane is depolarized (Akaike and Kaneda 1989; Legendre and Korn 1995). Moreover, changes with voltage of GABAergic IPSCs duration are characterized by a modification of the relative amplitude of the two decay components, whereas their decay time constants remain unchanged (Mellor and Randall 1998). This is the opposite for GlyRs. Voltage-dependent kinetics of glycine-gated channels A voltage dependence of transition rate constants for GABAA receptors, which might underlie changes in GABAergic mIPSC duration, has not yet been demonstrated (Mellor and Randall 1998), but the deactivation time course of GABAevoked responses and the GABAA receptors desensitization depend crucially on GABAA subunits combination (McClellan and Twyman 1999). This renders kinetic analysis with Markov model approximations much more difficult. However, the number of potential GlyR subunit combinations is much less than for GABA. By focusing my analysis on one type of GlyRs, presumably a1/b-like GlyRs (Legendre 1997), I was able to determine a Markov model describing GlyRs activation kinetics (Legendre 1998) and so could determine which GlyR gating reaction possessed a voltage dependence. The gating scheme I used provides good approximations of the activation and deactivation behavior of GlyRs receptors activated by short glycine applications over a wide range of agonist concentration, although no desensitized states were included (Legendre 1998). The desensitized states were not incorporated because they are too slow to influence the mIPSCs time courses at all voltages tested. Changes in the mean open time with voltage might, however, reflect open channel block mechanisms. But this cannot account for the change in glycine-evoked responses duration with voltage. The current-voltage (I-V) curve is linear in the voltage range over which changes in decay time duration occurs. Morever, single-channel conductance is insensitive to voltage for Vh between 250 and 120 mV (Legendre and Korn 1994). Finally it is unlikely that the agonist itself can block the glycine-gated channel as proposed for acetylcholine on the nicotinic receptors (Liu and Dilger 1991; Ogden and Colquhoun 1985; Sine et al. 1990; Sine and Steinbach 1984) because glycine is weakly charged at neutral pH. A decrease in the closing rate constant implies that opening of the GlyRs chloride channels is dependent on the membrane voltage only. This differs from most voltage-gated channels where typically all gating rates depend on voltage (Chen and Hess 1990; Horn and Vandenberg 1984; Keynes 1994; Kuo and Bean 1994), whereas closing rates can be voltage independent as for the Shaker potassium channel, the squid sodium channel or the N-type calcium channel (Aldrich and Stevens 1987; Boland and Bean 1993; Cota and Armstrong 1989; Miller 1990; Vandenberg and Bezanilla 1991). Mechanisms underlying the voltage dependence of the GlyRs closing rate constant Changes in closing rate constants with voltage have also been reported for acetylcholine receptors (AChRs) (Ascher et al. 1978; Auerbach et al. 1996; Colquhoun and Sakmann 1985; Magleby and Stevens 1972; Marchais and Marty 1979; Neher and Sakmann 1976; Sheridan and Lester 1977; Sine et al. 1990). Two types of mechanisms have been proposed to explain this voltage dependence for AChRs (Auerbach et al. 1996; Marchais and Marty 1979). The first mechanism is related to ions permeation through the pore of the channel. It implies that favored binding of permeant cations on its binding Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. VOLTAGE-DEPENDENT KINETICS OF THE GLYCINE RECEPTOR 2127 channel cannot close until the ion dissociates from its binding sites. A second mechanism, not entirely incompatible with the first, is related to voltage-sensitive charge movements in the protein during gating and has been proposed to explain changes in closing rate constants of mouse AChRs (Auerbach et al. 1996). It supposes that charged moieties in the AChRs protein change their disposition after agonist binding and move with respect to the electrical field when the channel gates (Auerbach et al. 1996). Both models could explain the voltage dependence of the GlyRs channel closing rate constants. The GlyRs channel pore has at least two anion binding sites (Bormann et al. 1987), and this binding could be favored at depolarized membrane potential, which will in turn hinder channel closing. But if the anion binding hypothesis is true, receptor channels with closely identical pore should have similar voltage-dependent properties. This is not the case for GlyRs and GABAA receptors (GABAAR). Both these receptors have highly conserved M2 domains that form the pore of the channel (Betz 1992; David-Watine et al. 1999), and the walls of the channels have very similar electrical properties (Bormann et al. 1987). Although responses evoked by fast applications of GABA increase in duration with voltage, even when fast desensitization processes are not involved, this is not due to a change in the deactivation time constant with voltage but to an increase in the relative proportion of the slow deactivation component (Mellor and Randall 1998). This is the opposite to what I observed for GlyRs, suggesting that changes in anion binding with voltage may not significantly modify the opening duration of the GlyR and GABAAR anionic channel. It is the therefore tempting to speculate that the decrease in the closing rate constant of GlyRs at depolarized potential results largely from charge movement with respect to the electrical field during channel gating. Studies using recombinant GlyRs having mutations in the pore region of the channel are needed to address this issue definitively (Auerbach et al. 1996). Physiological significance FIG. 6. Theoretical onset of responses evoked by step applications of 0.1 mM glycine. The rate parameters used to generate theoretical traces were kon 5 5 mM21s21, koff 5 1,550 s21, a1 5 620 s21, b1 5 8,938 s21, a2 5 1,300 s21, b2 5 3,180 s21, d 5 1,300 s21, and r 5 120 s21. A: example of theoretical traces obtained with the Markov model shown in Fig. 5A, with a1 and a2 being voltage dependent. B: analysis of the predicted change in the activation time constants with holding potential. As in Fig. 5, 2 models were compared with experimental data, where either the closing rate constants (●) or either the dissociation rate constant (E) were assumed to be voltage dependent. Note that only the model with voltage-dependent a accurately predicts my experimental data. C: predicted change in the relative proportion of the 2 onset components with voltage. The 2 models tested both fit well my experimental data and predict little change in the relative amplitude of the 2 rise phase components of the outside-out responses evoked by 0.1 mM concentration steps of glycine. site at more hyperpolarized potential will hinder channel closing and therefore increase the mean open time of the channel (Marchais and Marty 1979). This hypothesis supposes that the A slow desensitization process of GlyRs cannot play a significant role after release of a single vesicle, although it might modulate glycinergic synaptic efficacy when a longlasting (2– 4 s) depolarization of postsynaptic membrane is coupled with high-frequency inhibitory cell activity. In contrast, changes in the decay time of glycine evoked responses with voltage can enhance the efficacy of single inhibitory responses in the face of an increased excitation (Faber and Korn 1987). This implies that the increase of glycinergic inhibitory postsynaptic potential (IPSP) duration with membrane depolarization will significantly favor their summation or prolong the membrane hyperpolarization. This will, however, depend on the membrane time constant of the cell. Effectively, a membrane time constant larger than the deactivation time constant of the synaptic current will tend to reduce the effect of changes in the current decay time with voltage on IPSP duration but will increased its efficacy to control IPSP amplitude (Singer et al. 1998). Glycinergic synapses can also inhibit cell activity by shunting electrotonic transmission due to the evoked decrease in the cell input resistance. In this case, a depolarization of the membrane will also enhance the effect of glycinergic synapses on the input resistance of the cell, which Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. 2128 P. LEGENDRE in turn will increase the effectiveness in opposing excitatory electrotonic transmission. I thank Dr. Richard Miles for valuable help and discussions. This work was supported by Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Association Francaise contre les Myopathies. Address for reprint requests: P. Legendre, Institut des Neurosciences, Bat B. 6eme étage, boite 8, Université Pierre et Marie Curie, 7 Quai Saint Bernard, 75252 Paris Cedex 05, France. Received 22 April 1999; accepted in final form 16 June 1999. REFERENCES AKAIKE, N. AND KANEDA, M. Glycine-gated chloride current in acutely isolated rat hypothalamic neurons. J. Neurophysiol. 62: 1400 –1409, 1989. ALDRICH, R. W. AND STEVENS, C. F. Voltage-dependent gating of single sodium channels from mammalian neuroblastoma cells. J. Neurosci. 7: 418 – 431, 1987. ASCHER, P., MARTY, A., AND NEILD, T. O. Life time and elementary conductance of the channels mediating the excitatory effects of acetylcholine in Aplysia neurones. J. Physiol. (Lond.) 278: 177–206, 1978. AUERBACH, A., SIGURDSON, W., CHEN, J., AND AKK, G. Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di-, mono-, and unliganded receptors. J. Physiol. (Lond.) 494: 155–170, 1996. BEAN, B. P. Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51: 367–384, 1989. BETZ, H. Structure and function of inhibitory glycine receptors. Q. Rev. Biophys. 25: 381–394, 1992. BOLAND, L. M. AND BEAN, P. Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone-releasing hormone: kinetics and voltage dependence. J. Neurosci. 13: 516 –533, 1993. BORMANN, J., HAMILL, O. P., AND SAKMANN, B. Mechanism of anion permeation through channels gated by glycine and g-aminobutyric acid in mouse cultured spinal neurones. J. Physiol. (Lond.) 385: 243–286, 1987. BURGARD, E. C., TIETZ, E. I., NEELANDS, T. R., AND MACDONALD, R. L. Properties of recombinant gamma-aminobutyric acid A receptor isoforms containing the alpha 5 subunit subtype. Mol. Pharmacol. 50: 119 –127, 1996. CHEN, C. F. AND HESS, P. Mechanism of gating of T-type calcium channels. J. Gen. Physiol. 96: 603– 630, 1990. COLLINGRIDGE, G. L. AND LESTER, R.A.J. Excitatory amino-acid receptors in the vertebrate central nervous system. Pharmacol. Rev. 40: 143–210, 1989. COLQUHOUN, D. AND SAKMANN, B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. (Lond.) 369: 501–557, 1985. COTA, G. AND ARMSTRONG, C. M. Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent. J. Gen. Physiol. 94: 213–232, 1989. CURUTCHET, P., BOCHET, P., DE CARVALHO, L. P., LAMBOLEZ, B., STINNAKRE, J., AND ROSSIER, J. In the GluR1 glutamate receptor subunit a glutamine to histidine point mutation suppresses inward rectification but not calcium permeability. Biochem. Biophys. Res. Comm. 182: 1089 –1093, 1992. DAVID-WATINE, B., GOBLET, C., DE SAINT JEAN, D., FUCILE, S., DEVIGNOT, V., BREGESTOVSKI, P., AND KORN, H. Cloning, expression and electrophysiological characterization of glycine receptor alpha subunit from zebrafish. Neuroscience 90: 303–317, 1999. DERKACH, V., SURPRENANT, A., AND NORTH, R. A. 5-HT3 receptors are membrane ion channels. Nature 339: 706 –709, 1989. DOMINGUEZ-PERROT, C., FELTZ, P., AND POULTER, M. O. Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance. J. Physiol. (Lond.) 497: 145–159, 1996. ELMSLIE, K. S. AND JONES, S. W. Concentration dependence of neurotransmitter effects on calcium current kinetics in frog sympathetic neurones. J. Physiol. (Lond.) 48: 35– 46, 1994. ELMSLIE, K. S., ZHOU, W. AND JONES, S. W. LHRH and GTP-gamma-S modify calcium current activation in bullfrog sympathetic neurons. Neuron 5: 75– 80, 1990. FABER, D. S. AND KORN, H. Voltage-dependence of glycine-activated Cl2 channels: a potentiometer for inhibition? J. Neurosci. 7: 807– 811, 1987. FRANKE, C., HATT, H., AND DUDEL, J. Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle. Neurosci. Lett. 77: 199 –204, 1987. GUNDERSEN, C. B., MILEDI, R., AND PARKER, I. Properties of human brain glycine receptors expressed in Xenopus oocytes. Proc. R. Soc. Lond. B. Biol. Sci. 221: 235–244, 1984. GUNDERSEN, C. B., MILEDI, R., AND PARKER, I. Voltage-dependence of human brain glycine receptor-channels in Xenopus oocytes (Abstract). J. Physiol. (Lond.) 377: 40P, 1986. HAMILL, O. P., MARTY A., NEHER, E., SAKMANN, B., AND SIGWORTH, F. J. Improved patch clamp techniques for high-resolution current recordings from cells and cell free patches. Pflügers Arch. 391: 85–100, 1981. HATTA, K. AND KORN, H. Physiological properties of the Mauthner system in the adult zebrafish. J. Comp. Neurol. 395: 493–509, 1998. HORN, R. AND VANDENBERG, C. A. Statistical properties of single sodium channels. J. Gen. Physiol. 84: 505–534, 1984. JONES, M. V. AND WESTBROOK, G. L. Desensitized states prolong GABAA channel responses to brief agonist pulses. Neuron 15: 181–191, 1995. KEYNES, R. D. Bimodal gating of the Na1 channel. Trends Neurosci. 17: 58 – 61, 1994. KUO, C. C. AND BEAN, B. P. Na1 channels must deactivate to recover from inactivation. Neuron 12: 819 – 829, 1994. LEGENDRE, P. Pharmacological evidences for two types of postsynaptic glycinergic receptors on 52-hour old zebrafish larvae J. Neurophysiol. 77: 2400 –2415, 1997. LEGENDRE, P. A reluctant gating mode of glycine receptor channels determines the time course of inhibitory miniature synaptic events in zebrafish hindbrain neurons. J. Neurosci. 18: 2856 –2870, 1998. LEGENDRE, P. AND KORN, H. Glycinergic inhibitory synaptic currents and related receptor-channel in the Zebrafish brain. Eur. J. Neurosci. 6: 1544 – 1557, 1994. LEGENDRE, P. AND KORN, H. Voltage dependence of conductance changes evoked by glycine release in the zebrafish brain. J. Neurophysiol. 73: 2404 –2412, 1995. LESTER, R. A., CLEMENTS, J. D., WESTBROOK, G. L., AND JAHR, C. E. Channel kinetics determine the time course of NMDA receptor–mediated synaptic currents. Nature 346: 565–567, 1990. LIU, Y. AND DILGER, J. P. Opening rate of acetylcholine receptor channels. Biophys. J. 60: 424 – 432, 1991. MACONOCHIE, D. J. AND KNIGHT, D. E. A method for making solution changes in the submillisecond range at the tip of a patch pipette. Pflügers Arch. 414: 589 –596, 1989. MAGLEBY, K. L. AND STEVENS, C. F. The effect of voltage on the time course of end-plate currents. J. Physiol. (Lond.) 223: 151–171, 1972. MARCHAIS, D. AND MARTY, A. Interaction of permeant ions with channels activated by acetylcholine in Aplysia neurones. J. Physiol. (Lond.) 297: 9 – 45, 1979. MAYER, M. L., WESTBROOK, G. L., AND GUTHRIE, P. B. Voltage-dependent block by Mg21 of NMDA responses in spinal cord neurones. Nature 309: 261–263, 1984. MCCLELLAN, A. M. AND TWYMAN, R. E. Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors. J. Physiol. (Lond.) 515: 711–727, 1999. MELLOR, J. R. AND RANDALL, A. D. Voltage-dependent deactivation and desensitization of GABA responses in cultured murine cerebellar granule cells. J. Physiol. (Lond.) 506: 377–390, 1998. METCALFE, W. K., MENDELSON, B., AND KIMMEL, C. B. Segmental homologies among reticulospinal neurons in the hindbrain of the zebrafish larva. J. Comp. Neurol. 251: 147–159, 1986. MILLER, C. Annus Mirabilis of potassium channels. Science 252: 1092–1096, 1990. MULLE, C. AND CHANGEUX, J.-P. A novel type of nicotinic recepetor in the rat central nervous system characterized by patch-clamp techniques. J. Neurosci. 10: 169 –175, 1990. NEHER, E. AND SAKMANN, B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260: 799 – 802, 1976. NOWAK, L. M., BREGESTOVSKI, P., ASCHER, P., HERBET, A., AND PROCHIANTZ, A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462– 465, 1984. OGDEN, D. C. AND COLQUHOUN, D. Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction. Proc. R.. Soc. Lond. B. Biol. Sci. 225: 329 –355, 1985. OTIS, T. S. AND MODY, I. Modulation of decay kinetics and frequency of GABAA receptor–mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons. Neuroscience 49: 13–32, 1992. ROTHMAN, S. M. AND CHOI, D. W. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 13: 171–182, 1990. Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022. VOLTAGE-DEPENDENT KINETICS OF THE GLYCINE RECEPTOR SHERIDAN, R. E. AND LESTER, H. A. Rates and equilibria at the acetylcholine receptor of Electrophorus electroplaques: a study of neurally evoked postsynaptic currents and of voltage-jump relaxations. J. Gen. Physiol. 70: 187–219, 1977. SINE, S. M., CLAUDIO, T., AND SIGWORTH, F. J. Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities. J. Gen. Physiol. 96: 395– 437, 1990. SINE, S. M. AND STEINBACH, J. H. Agonists block currents through acetylcholine receptor channels. Biophys. J. 46: 277–283, 1984. 2129 SINGER, J. H., TALLEY, E. M., BAYLISS, D. A., AND BERGER, A. J. Development of glycinergic synaptic transmission to rat brain stem motoneurons. J. Neurophysiol. 80: 2608 –2620, 1998. STUART, G. J. AND REDMAN, S. J. Voltage dependence of Ia reciprocal inhibitory currents in cat spinal motoneurones. J. Physiol. (Lond.) 420: 111–125, 1990. VANDENBERG, C. A. AND BEZANILLA, F. Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon. Biophys. J. 60: 1499 –1510, 1991. YOON, K. W. Voltage-dependent modulation of GABAA receptor channel desensitization in rat hippocampal neurons. J. Neurophysiol. 71: 2151–2160, 1994. Downloaded from journals.physiology.org/journal/jn (184.072.149.166) on June 14, 2022.