Institut des Neurosciences, Université Pierre et Marie Curie, 75252 Paris Cedex 05, France
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ABSTRACT |
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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
outside-out current evoked by short applications (1 ms) of a saturating
concentration of glycine (3 mM). When the outside-out patch was
depolarized from 50 to +20 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
50 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 ± 2.8% of the total
current at
50 mV and 54.1 ± 10% at +20 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
70 mV and 0.22 ms at +20 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.
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INTRODUCTION |
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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
Mg2+ for N-methyl-D-aspartate
(NMDA) receptors (Mayer et al. 1984
; Nowak et al.
1984
) or by the neurotransmitter itself, as shown 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 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.
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METHODS |
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Isolated intact brain preparation
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
plasma-thrombin 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.
Outside-out patch-clamp recordings
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). Patch-clamp
electrodes were pulled from thick-wall (10-15 M
) 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 outside-out patches ranged from 2 to 10 G
.
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).
Drug delivery
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 µm. 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 µm (patch electrode resistance >10 M
) and a
diffusion coefficient for glycine close to 0.5-1 × 10
5
cm2s
1, the estimated
absolute exchange time was found to be
0.1 ms.
Outside-out patch current analysis
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
).
Kinetic modeling programs
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 ± SD
throughout unless otherwise noted.
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RESULTS |
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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
1 and heteromeric-like
/
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.
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 (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 =
50 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 60 to +20 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 = 10) when
Vh was increased from
50 to +20 mV
(Fig. 1). For
Vh less than
50 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
).
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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 fast = 5.1 ± 0.53 ms and
slow = 41.4 ± 7.8 ms (n = 10) at Vh =
50 mV. When the patches were depolarized to +20 mV,
fast and
slow
significantly increased to reach 9.8 ± 0.97 ms and 67.7 ± 11.5 ms (mean ± SD, n = 8), respectively (paired
t-test, P = 0.01).
fast increased progressively with voltage
(Fig. 2B). The relationship between
fast and the holding potential can be fitted by a single exponential function between
50 and +20 mV given an
increase in
fast with a limiting slope of
e-fold/95 mV (Fig. 2C).
slow also increased progressively with voltage
(Fig. 3A). Assuming that
slow also
changed exponentially when membrane potential was increased, we found
that
slow increased e-fold per 111 mV/between
50 and 10 mV. This is closely similar to that obtained for
fast.
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In contrast to the decay time constants, the relative amplitude of
these two decay components was not significantly voltage dependent
(paired t-test, P = 0.1). At
Vh = 50 mV, the fast decay component
represented 64.2 ± 2.8% (n = 10) of the total
current while depolarizing the patch to +20 mV slightly decreased its relative amplitude to 54.1 ± 10% (n = 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 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 (1 and
1 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
on [
on
=1/(
1 +
1)] with voltage can give information on the voltage
sensitivity of these rate constants. But, as the opening rate constant
1 of GlyRs is >10 times faster than the closing rate constant
1
(
1
9,000 s
1;
1
600-700
s
1) (Legendre 1998
), any
modifications in the onset duration will mainly reflect fluctuations in
the opening rate constant
1. For example a two time change in
1
will modify
on by
4% only.
To estimate the voltage dependence of the opening rate constant 1, 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 =
70 mV (0.2 ± 0.012 ms; n = 6) or
Vh =
50 mV (0.19 ± 0.01 ms;
n = 6) was not significantly modified (paired
t-test, P = 0.1) when the outside-out
patches were depolarized to 20 mV (0.22 ± 0.014 ms;
n = 8). It is therefore unlikely that the opening rate
constant
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 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
60 mV to +20
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
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The relative amplitude of these two components was concentration
dependent (Legendre 1998) but did not change
significantly with voltage (paired t-test, P = 0.1; Fig. 4D). For example,
on1 had a relative amplitude of 0.619 ± 0.13 at
Vh =
50 mV and 0.624 ± 0.15 at
Vh = +20 mV (n = 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
My experimental data therefore suggests that the closing rate
constants 1 and
2 (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 =
50 mV (see Fig.
5).
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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 50 and +20 mV
might also result from a slight voltage sensitivity of the dissociation
rate 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
and the rate constant d between A2C and A2C*
[N = 1 + (
1/d + 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
2 and the reverse rate constant
r between A2C* and A2C [N = 1 + (
2/r)]. Finally, a model in which
the opening rate constants
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
50 mV to +20 mV. This was not
experimentally observed (Fig. 3, C and D).
The first model had two voltage-dependent closing rate constants (1
and
2) with similar voltage sensitivities, as the two deactivation
components
fast and
slow were increased to the same extent by
depolarizing the patch to +20 mV (Figs. 2 and 3). Changes in the rate
constants
with voltage were calculated using the relation of the
form
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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
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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 60 to +20 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
was made to be
voltage dependent. Changes in
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 +20 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 = +20 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 compared with theoretical traces obtained from models 1 and 2. As previously mentioned, setting 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
60 to +20
mV, the two models predict that the relative proportion of the two
activation phase components will be little affected by 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
1 and
2,
the other gating reactions being voltage insensitive.
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DISCUSSION |
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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
The peak current evoked by a brief application of a saturating
concentration of glycine varied linearly between 60 and +20 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 =
50 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 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
GABA-evoked 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
1/
-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 50 and +20 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 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 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
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 long-lasting (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 in turn will increase the effectiveness in opposing excitatory
electrotonic transmission.
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ACKNOWLEDGMENTS |
---|
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.
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FOOTNOTES |
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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.
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.
Received 22 April 1999; accepted in final form 16 June 1999.
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REFERENCES |
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