Institut National de la Santé et de la Recherche Médicale U432, Neurobiologie et Développement du Système Vestibulaire, 34095 Montpellier cedex 5, France
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ABSTRACT |
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Chabbert, C.,
J. M. Chambard,
A. Sans, and
G. Desmadryl.
Three Types of Depolarization-Activated Potassium Currents in
Acutely Isolated Mouse Vestibular Neurons.
J. Neurophysiol. 85: 1017-1026, 2001.
The nature and
electrophysiological properties of
Ca2+-independent depolarization-activated
potassium currents were investigated in vestibular primary neurons
acutely isolated from postnatal mice using the whole cell configuration
of the patch-clamp technique. Three types of currents were identified.
The first current, sensitive to TEA
(ITEA) and insensitive to
4-aminopyridine (4-AP), activated at 40 mV and exhibited slow
activation (
ac, 38.4 ± 7.8 ms
at
30 mV, mean ± SD).
ITEA had a half activation potential
[Vac(1/2)] of
14.5 ± 2.6 mV
and was inactivated by up to 84.5 ± 5.7% by 10-s conditioning
prepulses with a half inactivation potential [Vinac(1/2)] of
62.4 ± 0.2 mV. The second current, sensitive to 4-AP (maximum block around 0.5 mM)
and to
-dendrotoxin (IDTX) appeared
at
60 mV. Complete block of IDTX was
achieved using either 20 nM
-DTX or 50 nM margatoxin. This current
activated 10 times faster than ITEA
(
ac, 3.5 ± 0.8 ms at
50 mV)
with Vac(1/2) of
51.2 ± 0.6 mV, and inactivated only slightly compared with ITEA (maximum inactivation, 19.7 ± 3.2%). The third current, also sensitive to 4-AP (maximum block at
2 mM), was selectively blocked by application of blood depressing
substance (BDS-I; maximum block at 250 nM). The BDS-I-sensitive
current (IBDS-I) activated around
60
mV. It displayed fast activation
(
ac, 2.3 ± 0.4 ms at
50 mV)
and fast and complete voltage-dependent inactivation.
IBDS-I had a
Vac(1/2) of
31.3 ± 0.4 mV and
Vinac(1/2) of
65.8 ± 0.3 mV.
It displayed faster time-dependent inactivation and recovery from
inactivation than ITEA. The three
types of current were found in all the neurons investigated. Although
ITEA was the major current, the
proportion of IDTX and
IBDS-I varied considerably between neurons. The ratio of the density of
IBDS-I to that of
IDTX ranged from 0.02 to 2.90 without
correlation with the cell capacitances. In conclusion, vestibular
primary neurons differ by the proportion rather than the type of the
depolarization-activated potassium currents they express.
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INTRODUCTION |
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Vestibular primary neurons are
involved in transmitting afferent information about accelerations from
the inner ear vestibular mechanoreceptors to vestibular nuclei.
Vestibular afferents are classified as regularly or irregularly
discharging patterns (Smith and Goldberg 1986).
Irregular afferents have phasic response dynamics, higher sensitivities
to natural and to external galvanic stimulations, and larger axons than
regular fibers. They also differ in their response to the activation of
efferent pathway (Goldberg et al. 1984
; Smith and
Goldberg 1986
). These authors suggest that discharge regularity
as well as the other functional properties of vestibular primary
afferents could be the consequence of differences in membrane conductances between each class of neurons. However, the intrinsic properties supporting these differences are unknown. We therefore developed a preparation of vestibular ganglion neurons acutely isolated
from mice. Previous analysis of voltage-activated conductances showed
the presence in all vestibular neurons of one sodium current (Chabbert et al. 1997
) and five calcium currents
(Chambard et al. 1999
; Desmadryl et al.
1997
), but potassium currents that might influence their
discharge patterns were not investigated yet. Since a large variety of
voltage-activated K+ currents have been described
in various neuronal preparations, and since they have been reported to
play an important role in shaping membrane electrical activity
(Rudy 1988
), we characterized these currents in
vestibular primary neurons.
Over the last two decades many studies have been performed,
characterizing the outward voltage-activated K+
conductances expressed in dorsal root ganglion (DRG) neurons (Everill et al. 1998; Gold et al. 1996
;
Kostyuk et al. 1981
; Robertson and Taylor
1986
), and other sensory neurons (Brew and Forsythe 1995
; Garcia-Diaz 1999
; Locke and
Nerbonne 1997a
; Manis and Marx 1991
;
McFarlane and Cooper 1991
; Oyelese and Kocsis
1996
; Rathouz and Trussell 1998
;
Stansfeld and Feltz 1988
). Identification of the
different current types according to their activation and inactivation
properties, which often overlap, remains difficult (Gold et al.
1996
; McFarlane and Cooper 1991
).
Pharmacological agents such as tetraethylammonium (TEA) and
4-aminopyridine (4-AP) have been widely used to separate different
classes of voltage-activated K+ currents.
However, separation of the different types of 4-AP-sensitive currents
has often been impaired both by the overlap in the doses required to
block each current (Gold et al. 1996
; Hoshi and
Aldrich 1988
; McFarlane and Cooper 1991
) and by
the voltage sensitivity of the blocking effect (Yeh et al.
1976
). Conversely, several peptide toxins that selectively
block each type of 4-AP-sensitive K+ current are
now available.
-Dendrotoxin (
-DTX), a toxin purified from snake
venom, has been reported to selectively block a fast activating and
partially inactivating 4-AP-sensitive K+ current
(Stansfeld and Feltz 1988
). More recently margatoxin (MgTX), a toxin purified from scorpion venom, has been shown to block a
4-AP-sensitive K+ current in human peripheral
T-lymphocytes (Garcia-Calvo et al. 1993
). Another
peptide purified from sea anemone, blood depressing substance (BDS-I),
has been reported to block a fast activating and inactivating
K+ current (Diochot et al. 1998
).
The aim of the present study was to identify the different types of depolarization-activated K+ currents present in vestibular primary neurons using these new pharmacological tools, and to characterize their respective kinetic properties. Records were obtained using the whole cell configuration of the patch-clamp technique applied to primary neurons acutely isolated from postnatal mice. Based on their pharmacological and electrophysiological properties, we report here for the first time that three distinct outward Ca2+-independent depolarization-activated K+ conductances are present in vestibular primary neurons. These three conductances are expressed in various proportions between neurons.
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METHODS |
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Cell culture
Potassium currents were studied in neurons acutely isolated from
the superior branch of the vestibular nerve innervating the utricular
macula and the horizontal and lateral cristae using an isolation
procedure previously described (Desmadryl et al. 1997).
Ganglia were aseptically dissected from postnatal day 5 to 8 (P5 to P8) mice (CERJ, Le
Genest, France) rapidly killed by decapitation (the day of birth was
considered as postnatal day 0). About 20 ganglia for each
experiment were collected in phosphate-buffered saline (PBS; Life
Technologies). We tested different incubation times and trypsin
concentrations without any difference in the amplitude or the shape of
the recorded currents. We settled on protocols employing trypsin at
0.25% for 12 min at 37°C in PBS containing 0.25% EDTA-trypsin (Life
Technologies). Ganglia were triturated with fire-polished Pasteur
pipettes of three decreasing diameters in a cell culture medium
containing Neurobasal medium (Life Technologies), 10% B27 (Life
Technologies), 25 µM glutamate, and 0.25 mM glutamine. Neurons were
plated onto 35-mm culture dishes (Nunc) coated with 10 µg/ml
poly-D-ornithine (Sigma) in cell culture medium. Cells were
used between 1 and 4 h after dissociation. Under phase contrast
microscopy, dissociated neurons had a spherical shape and birefringent
cytoplasm as previously reported (Desmadryl et al.
1997
). Cell diameters ranged between 12.5 and 25 µm. Only
isolated neurons exhibiting no processes were chosen for the
electrophysiological studies. Their capacitances ranged from 9 to 32 pF
(18.6 ± 4.4 pF, mean ± SD) in a sample of 80 neurons.
Electrophysiological recordings
Whole cell recordings of voltage-dependent potassium currents
were obtained at room temperature (25°C) under conditions optimized to ensure their complete isolation from other voltage-dependent currents. Tetrodotoxin (1 µM, Sigma) was used to block
Na+ currents, and extracellular
Na+ was replaced by choline. Calcium was omitted
from the extracellular medium, and 2 mM EGTA was added to block
voltage-activated Ca2+ currents, as well as
Ca2+-activated K+ currents.
The standard extracellular solution contained (in mM) 135 cholineCl, 5 KCl, 10 HEPES, 10 glucose, 1 MgCl2, 2 EGTA, and 0.001 TTX. For extracellular TEA solutions, cholineCl was replaced by
equimolar TEACl. The pH of the recording solutions was adjusted to 7.35 and osmolality set at 300 mOsm/l. Recording pipettes pulled from
hematocrit tubes (Modulohm I/S, Herlev, Denmark) were coated with ski
wax to reduce capacitive transients. Pipettes 2-3 M were filled
with the following intracellular solution (in mM): 135 KCl, 10 EGTA, 25 HEPES, and 10 glucose. The pH was adjusted to 7.35 and the osmolality
set at 300 mOsm/l. Whole cell currents were recorded using a Axopatch
200B (Axon Instruments, Foster City, CA) patch-clamp amplifier. After
seal formation and membrane disruption, cell capacitance and series
resistance were estimated from the decay of the capacitance transient
induced by a ±10-mV pulse from a holding potential (HP) of
100 mV.
Series resistance was in the range of 5-9 M
, and the membrane
capacitance could be charged with a time constant of 100 µs. Series
resistances were 85% compensated after cancellation of the capacitive
transients. No linear leakage compensation was performed. Voltage
errors resulting from uncompensated series resistances were corrected
only for the high-threshold TEA-sensitive current when maximum voltage error exceeded 5 mV. The liquid junction potential between the internal
and the extracellular solution, measured according to Neher
(1992)
, was
6.8 mV at 25°C for control extracellular
medium. Data presented are not corrected for junction potential unless specified. Current signals were filtered at 5 kHz, digitized, and stored.
Drugs
4-AP and TEA were obtained from Sigma-Aldrich and were dissolved
directly in the extracellular medium. -DTX (Latoxan), MgTX (Bachem),
and BDS-I (a generous gift from S. Diochot, Institut de
Pharmacologie Moléculaire et Cellulaire, Valbonne, France) were
dissolved as stock solutions at
10
4 M in bidistilled
water and stored at
80°C. Drugs and peptides were applied to the
bathing medium in the vicinity of the cell by a fast gravity perfusion system.
Analysis
All experimental parameters, such as the holding and test
potentials, were controlled with an IBM PC equipped with a Tecmar Labmaster analog interface (Axon Instruments). Cell stimulation, data
acquisition, and analysis were performed using Pclamp software (v 5.5 and v 6, Axon Instruments). The mean chord conductances were
calculated, assuming a potassium equilibrium potential of 71 mV. For
each component of the whole cell K+ current,
activation and inactivation curves of pooled data were best fitted with
single Boltzmann function of the form
G/Gmax = 1/{1 + exp[(V
V1/2)/k]}, where
V1/2 was half activation or inactivation potential [Vac(1/2),
Vinac(1/2)] and k the
limiting slope. Time constants (
) were best fitted with single
exponential function of the form A · exp(
t/
), where A is the current peak and
the exponential time constant. Except when specified, pooled data are
given as means ± SD. Statistical significances were examined using ANOVA, corrected for unequal variances when necessary.
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RESULTS |
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Whole cell voltage-activated outward K+ currents
K+ currents were evoked by depolarizing
pulses at various voltages from HP 100 mV. Figure
1 illustrates activation (Fig.
1A) and inactivation (Fig. 1B) of currents
recorded in representative neurons in control external solution.
Examination of their general shape suggested that they were composed of
at least two distinct components with an early transient component and
a sustained component in most neurons (Fig. 1Aa,
10 mV
trace). Analysis of the current-voltage (I-V) relationship
for the sustained component confirmed the presence of two distinct
currents. The first component of the sustained current activated at
potentials just positive to
60 mV, and the second above
30 mV. This
can be seen as an increase in the slope of the I-V curve
(Fig. 1Ab, arrow). The presence of two components in the
sustained current was further supported by plotting relative activation, expressed as
G/Gmax, versus test
potential in a sample of four neurons (Fig. 1Ac). The curve
was composed of two Boltzmann components (dotted lines) with
Vac(1/2) of
47 and
12 mV, which contributed to 45 and 55% of the total current, respectively, in this
cell. These two Vac(1/2) values
matched those estimated below for the
-DTX and TEA-sensitive
components.
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Steady-state inactivation of the whole cell K+
current confirmed that it was composed of different components. Figure
1Ba illustrates inactivation elicited at +20 mV by 10-s
conditioning prepulses to various voltages. Plotting current amplitudes
normalized to maximum current as a function of conditioning voltages
revealed that a large part of the whole cell K+
current did not inactivate even for long conditioning prepulses (Fig.
1Bb), whereas the inactivating portion exhibited distinct voltage dependence when measured at the beginning (), or the end
(
) of the test pulse. Plotting relative inactivation against conditioning voltages in a sample of five neurons indicated that the
early transient and sustained components inactivated with Vinac(1/2) of
76 and
61 mV, respectively.
To determine that depolarization-activated whole cell currents are
carried by K+, the reversal potential was
estimated by stepping the HP from 100 to
20 mV for 20 ms to
activate the whole cell K+ current, followed by
100-ms depolarizations from
100 to +20 mV in 10 mV increments.
Reversal potential Erev was estimated by measuring tail currents 2 ms after the end of the depolarizing pulses in external solutions containing 5 or 30 mM
K+. This procedure gave a mean
Erev of
71.2 ± 1.4 mV
(n = 8) in standard external solution and
32.7 ± 1.2 mV (n = 8) in 30 mM K+
external solution (including the junction potential values). The
calculated equilibrium potential in our recording conditions using the
Nernst equation gave
83.2 and
38 mV, respectively. From our data it
appears that the Erev of the whole
cell K+ current was dependent on the
K+ gradient, but less than that expected for an
absolute K+ current, suggesting that
K+ channels in our preparation are not absolutely
selective to K+. Alternatively, these
discrepancies could be the consequence of K+
accumulation in the extracellular space, as reported by Rathouz and Trussell (1998)
in neurons of the avian nucleus magnocellularis.
Pharmacological identification of the different components of the whole cell K+ current
We first studied the effect of external applications of increasing
concentrations of 4-AP and TEA on the whole cell
K+ current evoked in primary vestibular neurons.
Figure 2 illustrates the separation into
three distinct components of whole cell K+
current elicited in a representative neuron by depolarizing pulses to
10 mV (Fig. 2A) and on the corresponding I-V
relations (Fig. 2B). Applications of 4-AP at concentrations
below 0.5 mM (typically we used 0.1 mM) blocked a fast activating
sustained component of the whole cell K+ current
(Fig. 2, Aa and Ab, and C), that
activated from
60 mV (Fig. 2, Ba and Bb). Above
0.5 mM, 4-AP also blocked a fast activating transient component with
maximum effect around 2 mM (Fig. 2, Aa and Ac,
and C). This component of the whole cell
K+ current activated at
60 mV. Applications of
TEA blocked a component of the whole cell K+
current with an effect from 1 mM, and maximum block around 40 mM (Fig.
2C). The current sensitive to TEA
(ITEA) exhibited slow activation and
higher threshold of activation than the 4-AP-sensitive currents
(around
40 mV; Fig. 2, Ba and Bb). In most
studies, 4-AP did not completely block one component of the whole cell K+ current without affecting the other. We
therefore studied the effect of
-DTX, MgTX, and BDS-I, three
toxins reported to selectively block distinct 4-AP-sensitive
K+ currents. Figure
3 illustrates representative effects of
these toxins on single traces of whole cell K+
currents. Application of
-DTX blocked a fast activating sustained component of the whole cell K+ current with
noticeable effect at 2 nM and maximum blocking effect around 20 nM
(Fig. 3A). MgTX blocked a component of the whole cell
K+ current with an effect from 10 nM and maximum
blocking effect around 50 nM (Fig. 3B). Applications of
nanomolar concentrations of BDS-I blocked a fast activating transient
component of the whole cell K+ current with
noticeable effect from 20 nM and maximum blocking effect around 250 nM
(Fig. 3C). When used at its maximum blocking concentration,
-DTX impeded the effect of subsequent application of 0.1 mM 4-AP
(Fig. 3D). A similar result was obtained with MgTX (Fig.
3E). Each of the two toxins also prevented the effect of the
other without preventing the effect of 2 mM 4-AP on the fast activating
transient component (Fig. 3F). Presence of 250 nM BDS-I in
the external solution did not prevent the blocking effect of
-DTX or TEA, whereas it prevented those of 2 mM of 4-AP
(Fig. 3G).
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Figure 4 illustrates the isolation of the
three components of whole cell K+ current using
-DTX, MgTX, or BDS-I on depolarizing pulses to
10 mV (Fig. 4,
Aa-Ca) and on the corresponding I-V relations (Fig. 4, Ab-Cb). Pharmacological properties of the toxins
reported in Fig. 3, and the similarity in the I-V relations
shown in Fig. 4 indicate that
-DTX, MgTX, and 4-AP (below 0.5 mM)
affect the fast activating sustained component of the whole cell
K+ current, thereafter referred as
IDTX, whereas BDS-I and 4-AP (at
millimolar concentrations) affect its transient component, thereafter
referred as IBDS-I.
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Characterization of ITEA
The high-threshold slow activating and inactivating TEA-sensitive
current was isolated by subtracting traces evoked in 40 mM TEA external
solution from those elicited in standard external solution (Fig.
5). Steady-state voltage-dependent
activation of ITEA was studied using
depolarizing pulses at various voltages from HP 100 mV.
ITEA appeared from a threshold between
50 and
40 mV and exhibited slow activation and slow time-dependent
inactivation (Fig. 5Aa). This current was fully activated
around +20 mV. Figure 5Ab illustrates the plot of relative
activation of ITEA expressed as
G/Gmax as a function of the
test potential in a sample of nine neurons. Values of current
amplitudes were taken 5 ms before the end of the test pulse. The data
curve was best fitted with single Boltzmann function with
Vac(1/2) of
14.5 ± 2.6 mV and
k of 10.2 ± 0.6 mV (means ± SE). The time
constant for activation (
ac) of
ITEA was voltage dependent and ranged
from 38.4 ± 7.8 ms at
30 mV, to 5.1 ± 1.7 ms at +25 mV
for a sample of seven neurons.
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The inactivation, not visible within the 150-ms pulse, was evident for
longer depolarizations. Steady-state voltage-dependent inactivation of
ITEA was studied on currents elicited
at +20 mV following 10-s prepulse protocol (Fig. 5Ba).
Current amplitudes measured 5 ms before the end of the test pulse were
normalized to maximum current and expressed as
I/Imax. In a sample of six neurons, the mean maximum inactivation of
ITEA was 84.5 ± 5.7%. Plot of
relative inactivation as a function of the prepulse voltages was best
fitted with single Boltzmann function (Fig. 5Bb), with Vinac(1/2) of 62.4 ± 0.2 mV,
with k of 11.2 ± 0.3 mV (means ± SE).
Steady-state time-dependent inactivation and recovery from inactivation
of ITEA were studied using two-step
protocols. Inactivation of ITEA
elicited by depolarizing pulses to +20 mV was achieved by increasing
the duration of conditioning prepulses to 20 mV (Fig.
5Ca). Recovery of ITEA
previously inactivated by holding the neuron at
20 mV for several
minutes was achieved by increasing the duration of conditioning
prepulses to
100 mV (Fig. 5Da). Relative time-dependent
inactivation (Fig. 5Cb) and recovery from inactivation (Fig.
5Db) of ITEA were plotted
as a function of prepulse duration in a sample of seven neurons. Data
points were fitted with single exponential functions of the form
A exp(
t/
), with time constants of 6.7 ± 1.0 s and 254.4 ± 14.1 ms (solid lines).
Steady-state voltage-dependent deactivation of
ITEA was determined using a protocol
in which a 20-ms pulse to +10 mV was applied to fully activate the
current, followed by a range of more negative potentials from 45 to
70 mV to shut it (not shown). The fit of tail currents revealed that
they decayed with a single exponential time course. The mean time
constant of deactivation (
deac) at
50 mV was 26.4 ± 3.6 ms for a sample of four neurons. The
presence of 20 nM
-DTX, 50 nM MgTX, or 2 mM 4-AP in the external
solution did not change either the maximum amplitudes at +20 mV
(Imax) nor the kinetic characteristics
of ITEA (Table
1).
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Characterization of IDTX
The low-threshold, fast activating, sustained -DTX-sensitive
current was isolated by subtracting traces evoked in the presence of 20 nM
-DTX from those elicited in control solution (Fig.
6). From HP
100 mV,
IDTX activates at a threshold between
70 and
60 mV, and was fully activated at around
30 mV (Fig.
6Aa). It exhibited fast activation and very little
time-dependent inactivation even for 10-s depolarizing pulses. Plot of
relative activation of IDTX as a
function of the test potential in a sample of seven neurons (Fig.
6Ab,
) was best fitted with single Boltzmann function with Vac(1/2) of
51.2 ± 0.6 mV, and k of 4.0 ± 0.6 mV (means ± SE). The rise
time of activation was voltage dependent and displayed very fast
kinetics. On a sample of seven neurons,
ac
ranged from 3.5 ± 0.8 ms at
50 mV to 1.4 ± 0.3 ms at
10
mV. The current showed weak voltage-dependent inactivation (Fig.
6Ba). A 10-s prepulse protocol inactivated the current
evoked at
10 mV by 19.7 ± 3.2% (n = 5; Fig.
6Bb). Increasing prepulse duration up to 30 s produced
a maximum inactivation of about 25%.
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Steady-state voltage-dependent deactivation was studied by applying a
10-ms pulse to 30 mV to fully activate
IDTX, then stepping down to more
negative potentials from
45 to
70 mV. In a sample of five neurons,
the mean
deac was 15.6 ± 3.1 ms at
50 mV.
The Imax values, and kinetic
characteristics of IDTX, did not
significantly differ when MgTX was substituted for -DTX (Table 1 and
Fig. 6, Ab and Bb,
).
Characterization of IBDS-I
The low-threshold, fast activating and fast inactivating BDS-I-sensitive current was isolated either by subtracting traces evoked in the presence of 250 nM BDS-I from control, or taking advantage of its fast and complete time-dependent inactivation property (see below and Fig. 7). Results obtained by the two procedures did not differ significantly.
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Steady-state voltage-dependent activation of
IBDS-I was studied by subtracting
traces elicited by 125-ms test pulses ranging from 80 to +10 mV from
HP
50 mV, from those obtained with a 200-ms prepulse to
100 mV.
IBDS-I was activated at a threshold between
60 and
50 mV, with a fast activation and fast inactivation kinetics as shown for a representative neuron (Fig. 7Aa).
IBDS-I was fully activated at 0 mV and
almost totally inactivated after 150 ms.
Activation of IBDS-I estimated at the
peak of the current in a sample of 15 neurons (Fig. 7Ab)
gave Vac(1/2) of 31.3 ± 0.4 mV
and k of 8.5 ± 0.4 mV (means ± SE). The rise
time of activation was of the same order as that of
IDTX.
ac and
decay time (
inac) were voltage dependent,
ranging from 2.3 ± 0.4 ms at
50 mV to 0.8 ± 0.1 ms at +10
mV, and 53.5 ± 11.7 ms at
50 mV to 12.2 ± 3.1 ms at +10
mV, respectively.
Steady-state voltage-dependent inactivation of
IBDS-I was studied using a 200-ms
pulse to 100 mV from HP
50 mV, followed by 100-ms conditioning
steps between
120 and
10 mV, before a 200-ms test pulse to +10 mV.
Traces for a representative neuron and the relative inactivation for a
sample of seven neurons are shown in Fig. 7, Ba and
Bb, respectively. IBDS-I had
Vinac(1/2) of
65.84 ± 0.3 mV,
and k of 5.51 ± 0.25 mV (means ± SE).
Time dependence of inactivation and recovery from inactivation of
IBDS-I were studied using two step
protocols in the presence of 20 nM -DTX and 20 mM TEA to reduce TEA-
and
-DTX-sensitive currents. Under these experimental conditions,
the Imax values and the kinetic
characteristics of IBDS-I did not
change (Table 1). IBDS-I evoked by
250-ms test pulses to 0 mV from HP
100 mV were progressively
inactivated by increasing the duration of a conditioning prepulse to
50 mV (Fig. 7Ca). After complete inactivation was achieved
by holding the membrane potential to
50 mV,
IBDS-I progressively recovered when
the duration of a conditioning prepulse to
100 mV was increased (Fig.
7Da). Time-dependent inactivation and recovery from
inactivation of IBDS-I estimated in a
sample of eight neurons had mean time constants of 25.4 ± 0.7 ms
(Fig. 7Cb) and 95.8 ± 4.0 ms (Fig. 7Db), respectively.
Distribution of K+ currents among vestibular primary neurons
The three types of depolarization-activated K+ current were found in all neurons investigated, with substantial variations in their relative amplitudes, densities, and distributions (Table 2). ITEA was the predominant current making up to 56% of the total current. There was a large variability in the relative expression of the two fast activating currents. The ratio of the density of IBDS-I and IDTX varied from 0.02 to 2.90 (mean 0.90 ± 1.20; n = 38). No correlation between the size or the capacitance of the recorded neurons and the relative distribution of each current was noticed (data not shown).
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DISCUSSION |
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By using a combination of pharmacological and electrophysiological
approaches, we identified and characterized for the first time, three
different types of Ca2+-independent
depolarization-activated K+ currents in
vestibular primary neurons acutely isolated from postnatal mouse.
Experimental conditions, previously used to study voltage-activated
Na+ and Ca2+ conductances
on the soma of neurons enzymatically dissociated from the vestibular
ganglion (Chabbert et al. 1997; Chambard et al.
1999
; Desmadryl et al. 1997
), were adapted to
the study of voltage-activated K+ currents. The
first current was isolated on the basis of its sensitivity to TEA and
was termed ITEA. Two fast activating
currents sensitive to 4-AP were isolated according to their sensitivity to two toxins,
-DTX and BDS-I, and were termed
IDTX and
IBDS, respectively.
ITEA
ITEA is characterized by its
sensitivity to TEA and its insensitivity to 4-AP in the same way that
IK described in a majority of DRG
neurons (Gold et al. 1996; Kostyuk et al.
1981
), in other sensory neurons (Garcia-Diaz
1999
; Manis and Marx 1991
; McFarlanne and
Cooper 1991
), and in a number of cortical neurons (for review see Locke and Nerbonne 1997a
),
ITEA exhibits a high threshold of
activation (between
30 and
40 mV), displays voltage-dependent activation, with a time course of activation 10 times slower than those
of the 4-AP-sensitive currents. Although its half activation potential
(
15 mV) is comparable to those found in adult rat DRG neurons
(IKlt in Gold et al.
1996
) and in neurons from the auditory brain stem nuclei
(Rathouz and Trussel 1998
), it differs notably from
several other sensory neurons. For example, its half activation potential is 30 mV more negative that in neonatal rat DRG neurons (McFarlane and Cooper 1991
), and 20 mV more negative
than in several cortical neurons (see Locke and Nerbonne
1997a
). Such a property is of importance regarding its putative
function in limiting firing by holding the membrane potential near
Ek with the consequence of inducing
rapid accommodation during sustained depolarization (Oyelese and
Kocsis 1996
). Although the majority of
IK in other sensory neurons
(Kostyuk et al. 1981
; McFarlane and Cooper
1991
; Rathouz and Trussel 1998
), and other cell
types (see Rudy 1988
) are usually described as
noninactivating currents, ITEA
displays voltage-dependent inactivation with a half inactivation
potential around
65 mV. Similar observations were previously reported
in rat visual cortical neurons (Locke and Nerbonne
1997a
), and in adult rat DRG neurons (Gold et al.
1996
). Gold et al. (1996)
showed that two out of
the three types of IK found in these
cells (termed Iki and
Iklt) displayed such properties. In
most respects, ITEA found in
vestibular neurons is identical to
Iklt described in those cells.
IDTX
This current shares most of the properties that define
ID (a -DTX-sensitive current) in
sensory neurons (Everill et al. 1998
; Stansfeld
and Feltz 1988
; Stansfeld et al. 1986
, 1987
),
namely its low-voltage threshold, fast time course of activation and deactivation, voltage-dependent activation, and partial steady-state inactivation. K+ currents possessing these
properties have been described in a wide variety of neurons
(Brew and Forsythe 1995
; Foehring and Surmeier
1993
; Rathouz and Trussel 1998
; Reid et
al. 1999
; Southan and Robertson 1998
;
Storm 1988
). In some preparations, discrepancies can be
noticed in the voltage dependence for activation (Locke and
Nerbonne 1997a
; McFarlane and Cooper 1991
;
Wu and Barish 1992
) and in the sensitivity to
-DTX
(Everill et al. 1998
; Wu and Barish 1992
). Such discrepancies could be the consequence of a lack of selectivity of
-DTX, when used over nanomolar concentration range, since other types of K+ channels have been
reported to be sensitive to
-DTX. For example, hippocampal neurons
express a population of IA channel
(Ficker and Heinemann 1992
) that possess a binding site
for
-DTX (Halliwell et al. 1986
), whereas
IA channels are usually reported to be
insensitive to
-DTX. In our experimental conditions, no effect of
-DTX on the kinetics or the voltage dependence of
IBDS was noticed. Similarly an
-DTX
sensitivity has been described for IK
in guinea pig DRG neurons (Penner et al. 1986
). This was
not the case in the present study. Another explanation for the
variability is that several types of
-DTX-sensitive
K+ currents, distinct from
IA or
IK are expressed in different neurons, and sometimes within a single neuron as reported in human peripheral myelinated neurons (Reid et al. 1999
). To determine
whether a single type or several populations of
IDTX are present in vestibular primary
neurons, it will be important to determine which
-subunits form the
IDTX K+
channels. The coupled sensitivities of
IDTX to
-DTX and MgTX we described
here is a first step on this way. Only three types of
-subunit,
Kv1.1, Kv1.2, and Kv1.6, have been reported to be sensitive to
-DTX,
and only Kv1.2 is insensitive to TEA (Grissmer et al.
1994
). Although this observation suggests that Kv1.2 is one of
the
-subunits that form the IDTX
channel in vestibular neurons, it cannot be excluded that Kv1.1 and
Kv1.6 may also be involved in the structure of this channel, since they
have been described as heteromeric structures with Kv1.2 in
K+ channels of the rat cerebellum (Koch et
al. 1997
). The involvement of
IDTX in limiting repetitive firing
both in sensory (Stansfeld et al. 1986
) and cortical
neurons (Brew and Forsythe 1995
; Wu and Barish
1992
) is widely accepted. However, the precise role of
ID in shaping a single action
potential remains somewhat uncertain. By directly measuring the action
potential-evoked Ca2+ rise in basket cell
terminals, Tan and Llano (1999)
indicated that
4-AP-sensitive K+ channels are involved in
depolarizing the action potential, but not the
-DTX-sensitive ones.
Opposite observations were made in rat visual cortical neurons, where
ID plays an important role in action
potential depolarization (Locke and Nerbonne 1997b
). Other authors suggest that ID might be
a large component of the afterhyperpolarization of action potential in
CA1 pyramidal neurons (Golding et al. 1999
), or rather
be involved in the control of resting membrane potential in basket
cells terminals (Robertson and Southan 1999
).
IBDS
IBDS shares most of the
properties previously reported for IA
in a wide variety of neurons (Rudy 1988).
IA is thought to modulate the timing
of repetitive action potential generation, the repolarization of single
action potential, and the time required to reach the threshold to fire
an action potential (Storm 1988
; Tan and Llano 1999
; Wu and Barish 1992
). A threshold for
activation around
60 mV, an absence of sensitivity to
-DTX, and
time constants for activation and inactivation in the same range to
those of IBDS-I have been described
for IA (rise time <0.5 ms; decay time
10-25 ms at 0 mV) in DRG neurons (Stansfeld et al.
1987
). Kinetics and voltage dependence for activation
and inactivation in the same range as those found for
IBDS-I were reported for
IA in cochlear ganglion neurons
(Garcia-Diaz 1999
) [
ac at 0 mV
<0.2 ms; Vac(1/2) =
38.4 mV;
Vinac(1/2) =
75 mV], and in nodose
neurons (McFarlane and Cooper 1991
)
[
ac at
10 mV = 1-1.5 ms and decay
time 10-30 ms; Vac(1/2) =
21 mV;
Vinac(1/2) =
73 mV].
IBDS-I found in vestibular neurons
displays a kinetic of recovery from inactivation slower than those
reported in dorsal cochlear nucleus pyramidal cells (Kanold and
Manis 1999
), and in basal ganglia and basal forebrain neurons
(Tkatch et al. 2000
), but faster than those reported in hippocampal neurons (Martina et al. 1998
; Wu and
Barish 1992
), and in striatal neurons (Song et al.
1998
). Attempts to identify the subunits that form
IA-type channels in other neurons
revealed that Kv 1.4, Kv 3.3 and Kv 3.4, Kv 4.2 and Kv 4.3
-subunits
underlie the transient K+ current (Diochot
et al. 1998
; Ohya et al. 1997
; Rudy et
al. 1999
). Analysis of the biophysical properties of the
current indicated that the time course of inactivation of
IA tends to be voltage independent
when carried by various Kv 4 channels, and highly voltage dependent, as
is the case for IBDS-I, when carried
by other subunits (Everill et al. 1998
). An interesting
observation is the fact that in our preparation,
IBDS-I displays a sensitivity to
nanomolar concentrations of BDS-I. This result is of interest first
because it constitutes the first demonstration of a BDS-I sensitivity
of a neuronal IA, and second because
it suggests the identity of at least one of the subunits that form the
channel, since the toxin isolated from sea anemone is a selective
blocker of the K+ channels form with Kv3.4
-subunit (Diochot et al. 1998
). Diochot et al.
(1998)
also reported a small blocking effect of BDS-I on the
Kv1.2 containing K+ channel expressed in
COS cells. It is unlikely, however, that IBDS-I is in fact an inactivating
component of the ID current since the
concentration of BDS-I we used was 40 times smaller than those used in
the COS-transfected cells. Again, alternative experimental approaches
will be needed to determine which
-subunits form the
IBDS-I channels present in vestibular
neurons, and whether they are assembled in heteromeric structures.
Our results demonstrate that the distribution of the three distinct
depolarization-activated K+ currents in
vestibular neurons is heterogeneous. Although
ITEA is the major current, the
proportion of IDTX and
IBDS-I varies considerably from one
neuron to another. Whether these observations reflect different
functional populations of neurons remains uncertain since the current
density ratios of
IBDS-I:IDTX
within neurons are distributed as a range rather than a distribution
into distinct groups. Moreover, there was no obvious correlation
between the size or the capacitance of the recorded cells and the
respective densities of the two 4-AP-sensitive currents. Similar
observations have been reported in rat DRG neurons (Everill et
al. 1998), where IK,
ID, and
IA are expressed with a large
variability between cells, and as a range rather than in distinct
sub-groups.
The role that each of the three K+ currents plays
in determining the electrophysiological properties of vestibular
neurons now needs to be correlated with the different firing patterns (regular and irregular), exhibited by the primary afferent
(Goldberg et al. 1984). The presence of the three types
of K+ currents within all the neurons
investigated and the large variability in the expression of the two
fast activating currents suggest that, if they are involved in shaping
action potential, a variation in their relative expression would be
sufficient to account for the diversity in the electrical activity
recorded in these neurons. The use of specific toxins should permit
determination of the involvement of each of the voltage-activated
K+ currents in the electrical activity of the
vestibular neurons. However, such investigations should be carefully
conducted, since these compounds could interact with other
voltage-activated ionic conductances (e.g., BDS-I has a slight effect
of on sodium current) (Diochot et al. 1998
).
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ACKNOWLEDGMENTS |
---|
The authors thank Prof. J. M. Goldberg and Dr. J. Ashmore for carefully reading the manuscript. We are grateful to G. Dayanithy, M. Desarménien, F. Scamps, J. Valmier, and G. Lennan for helpful discussions and comments on the manuscript. We also thank S. Diochot and L. Béress (Klinikum der Christian-Albrechts-Universität zu Kiel, Kiel, Germany) for providing BDS-I.
This work was supported by Division de la Recherche et des Etudes
DoctoralesUniversité Montpellier 2 to C. Chabbert and Centre
National des Etudes Spatiales Grant 99-793.
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FOOTNOTES |
---|
* C. Chabbert and J. M. Chambard contributed equally to this project.
Address for reprint requests: C. Chabbert, INSERM U432, Neurobiologie et Développement du Système Vestibulaire, UM2, cp 089 place E. Bataillon, 34095 Montpellier cedex 5, France (E-mail: chabbert{at}univ-montp2.fr).
Received 20 July 2000; accepted in final form 20 November 2000.
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REFERENCES |
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