Two types of voltage-dependent potassium channels in outer
hair cells from the guinea pig cochlea
Thierry
van den Abbeele1,2,
Jacques
Teulon2, and
Patrice Tran
Ba
Huy1
1 Laboratoire de Neurobiologie
des Systèmes Sensori-moteurs, Centre National de la Recherche
Scientifique, Unité Propre de Recherche et de l'Enseignement
Supérieur 7060, Faculté de Médecine
Lariboisière and 2 Institut
National de la Santé et de la Recherche Médicale U426,
Faculté de Médecine Xavier Bichat, Paris Cedex 18, France
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ABSTRACT |
Cell-attached and cell-free configurations of the patch-clamp
technique were used to investigate the conductive properties and
regulation of the major K+
channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major
voltage-dependent K+ channels. A
Ca2+-activated
K+ channel with a high conductance
(220 pS,
PK/PNa = 8) was found in almost 20% of the patches. The inside-out activity
of the channel was increased by depolarizations above 0 mV and
increasing the intracellular Ca2+
concentration. External ATP or adenosine did not alter the
cell-attached activity of the channel. The open probability of the
excised channel remained stable for several minutes without rundown and
was not altered by the catalytic subunit of protein kinase A (PKA)
applied internally. The most frequent
K+ channel had a low conductance
and a small outward rectification in symmetrical
K+ conditions (10 pS for inward
currents and 20 pS for outward currents, PK/PNa = 28). It was found significantly more frequently in cell-attached and
inside-out patches when the pipette contained 100 µM acetylcholine. It was not sensitive to internal
Ca2+, was inhibited by
4-aminopyridine, was activated by depolarization above
30 mV,
and exhibited a rundown after excision. It also had a slow inactivation
on ensemble-averaged sweeps in response to depolarizing pulses. The
cell-attached activity of the channel was increased when adenosine was
superfused outside the pipette. This effect also occurred with permeant
analogs of cAMP and internally applied catalytic subunit of PKA. Both
channels could control the cell membrane voltage of outer hair cells.
hair cells; potassium channels; delayed rectifier; calcium-activated channels
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INTRODUCTION |
MOST STUDIES ON K+
currents in mammalian outer hair cells (OHC) have used the whole cell
patch-clamp technique. Earlier reports stated that
Ca2+-dependent
K+ currents were predominant in
these cells (1, 13, 26), but recent studies indicate that
Ca2+-independent types of
K+ channels are important in
isolated OHC (18, 23) and in the isolated whole organ of Corti (19).
There is also controversy as to whether the acetylcholine-sensitive
K+ currents are associated with
metabotropic receptors (15) or ionotropic receptors (7,
12, 13), although the most recent electrophysiological (4, 23) and
molecular studies (6, 10) are in favor of ionotropic receptors. The ion
channels on the basolateral membrane of the OHC could play an important
role by regulating the cell voltage, because the electromotive response of OHC depends on the membrane potential. Only two studies have examined individual K+ channels on
the hair cell membrane of mammals. Ashmore and Meech (1) described two
types of Ca2+-activated
K+ channels: a high-conductance
one (240 pS) and a low-conductance one (40 pS). The presence of
high-conductance K+ channels was
subsequently confirmed by Gitter et al. (9), although their sensitivity
to internal Ca2+ and blockers was
not confirmed.
This study describes the single-channel properties of two types of
voltage-dependent K+ channels. The
first is Ca2+ dependent, has a
high conductance (near 220 pS) and the general properties of
maxi-K+ channels or
BKCa, and is regulated by
intracellular Ca2+ and voltage.
The second is Ca2+ insensitive,
has a low conductance (20 pS), and has many features of delayed
rectifier channels. It is more frequently found when acetylcholine is
present in the pipette and seems to be regulated by adenosine, cAMP,
and protein kinase A (PKA). These channels probably interact to control
the membrane potential of OHC.
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METHODS |
Tissue preparation.
Healthy pigmented guinea pigs (200-300 g, Preyer's reflex
positive) were killed with an overdose of pentobarbital sodium (30 mg/kg) and decapitated. Both bullas were quickly removed, the lateral
wall of the bony cochlea was removed under a binocular microscope, and
the second and third turns were dissected out into a Petri dish
containing Leibowitz L-15 medium. The organ of Corti was gently removed
using fine needles and transferred in a droplet of medium to a Petri
dish. OHC were mechanically isolated by gentle refluxing through a
100-µl Gilson pipette tip.
Measurements.
Isolated OHC were dissected out from the two middle turns of the
cochlea and placed in a chamber on the stage of an inverted microscope
(IM 35, Zeiss, Oberkochen, Germany) at room temperature (20-25°C). They were readily distinguished from inner hair
cells by their cylindrical shape and height (~40-70 µm). Any
OHC that showed loss of turgor, swelling, displacement of the nucleus
from the basal pole, or Brownian intracytoplasmic movements was
discarded. Single-channel currents were recorded from cell-attached and
excised, inside-out patches of basolateral membranes using the
patch-clamp technique (11). Currents were measured with an RK-400
patch-clamp amplifier (Biologic, Claix, France) and stored on a digital
audiotape recorder (DTR 1201, Biologic) for further analysis. The
pipette voltage was monitored with an IBM-compatible computer using a digital-to-analog converter and custom software (T. Van Den Abbeele). The current was low-pass filtered with an eight-pole Bessel filter (model 902 LPF2; Frequency Devices). Pipettes were made from
microhematocrit capillary tubes (CHR Badram, Bizkerod, Denmark) and
coated with Sylgard (Dow Corning, Seneffe, Belgium). When the pipette
solution was not a control NaCl solution, the leak current, which
partly reflects the occurrence of a liquid junction potential, was
zeroed electrically, but we took into account the fact that the
junction potential disappeared after sealing, unmasking the
compensating voltage (3). The membrane potential
(Vm), expressed
as the potential at the inner side of the membrane relative to the
pipette medium, is the algebraic sum of the spontaneous membrane
potential (Em)
and the clamp potential
(Vc =
Vpipette).
K+ currents from the inner to the
outer side of the membrane patch are given a positive sign and are
shown as upward deflections. The bath reference electrode was a 0.5 M
KCl (in 4% agar) bridge connected to an Ag-AgCl half-cell.
Data analysis.
For standard analysis, current records were low-pass filtered
(0.5-1 kHz), digitized (sampling rate 3 kHz) using an
IBM-compatible microcomputer with a Labmaster DMA card and the Biopatch
(Biologic) acquisition program, and further analyzed with custom
software. The channel activity
(NPo) or the
open-state probability
(Po) were
obtained from NPo = I/i,
where I is the mean current carried through the membrane patch by channels and
i is the unit current. The total
number of channels per patch (N) was
estimated by forming inside-out patches in a standard solution bath
containing 1 mM CaCl2 to maximally
activate Ca2+-activated channels
and then 10
9 M
CaCl2 to eliminate the activity of
Ca2+-activated channels. The
baseline was determined in excised patches after rundown or blockade by
barium or 4-aminopyridine (4-AP) in the specific case of the
low-conductance channels insensitive to
Ca2+. Digitized data points were
used to construct amplitude histograms; I was calculated as the integral of
the currents, using the closed level as reference. The current reversal
potential
(Einv) was estimated by fitting
i-V
relationships for each patch to a third-order polynomial function. The
relative permeabilities of the K+
channels,
PK/PNa,
were calculated with the following formulas derived from the
Goldman-Hogkin-Katz modified constant field equation
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where
ix is the
current carried by the ion X,
Px is the ion
permeability for X, and
[X]i
and
[X]o
are the intracellular and extracellular ion concentrations,
respectively, assuming that when E = Einv,
ix = 0 and
that the permeability of the channels for anions is negligible
The
Levenberg-Marcquardt algorithm (Sigmaplot 2.0., Jandel) was used to fit
the
Po/Vm
relationship to a Boltzmann distribution
where
P(0) is the resting
Po,
V50 is the
half-maximal voltage, and d is the
slope constant.
Kinetic analyses were performed only on patches containing one level of
activated channel, and current records were low-pass filtered (2 kHz)
and digitized (sampling rate, 5 kHz). Idealized recordings were
obtained by a half-amplitude threshold method, after analog filtering
to avoid that spontaneous peaks of the baseline exceeded the detection
threshold. Intervals were measured, binned, and fitted to the sum of
exponentials by the maximum likelihood method (Biopatch Analysis
Software, Biologic). The dwell-time distributions were fitted to the
following function
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where
N is the number of events,
A and
B are the coefficients, and
1 and
2 are the fast and slow time
constants, respectively.
Solutions.
The OHC were initially bathed in a standard solution containing (in mM)
140 NaCl, 4.8 KCl, 1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose,
adjusted to pH 7.4 with NaOH. The pipette solution usually contained
the same solution, except that 140 mM NaCl was replaced by 140 mM KCl
and the pH was adjusted to 7.4 with KOH. In another protocol, 100 µM
acetylcholine chloride was added to the pipette solution. Once a
cell-attached patch had formed, the cell was superfused with a stream
of solution from one of a series of pipette outlets. The cell-attached
configuration was usually stable under these circumstances, and the
flow of the liquid did not displace the cell. Tests with colored
solutions showed that the cell was rinsed in <5 s. The same
superfusion system was used after excision to produce the inside-out
configuration, but the control solution was a standard solution at pH
7.2 plus 1 mM CaCl2.
Ca2+ concentrations below
10
5 M were obtained by
adding EGTA and Ca-EGTA (20). The free
Ca2+ concentration was calculated
by an iterative method using stability constants for all reactions
between Ca2+,
H+,
Mg2+, and EGTA
(10
9 M was obtained by
adding 2 mM EGTA and 0 mM Ca-EGTA). The concentration of
Mg2+ was increased to 1.1 mM
MgCl2 for 0.1 mM ATP to compensate
for its chelation by ATP (24).
Ca2+ chelation was negligible at
this ATP concentration.
The catalytic subunit of PKA (from pig heart; Sigma) was dissolved in
NaCl standard solution with
10
9 M
CaCl2 by adding 2 mM EGTA. The
final solution contained 20 U/ml PKA, together with 0.1 mM ATP and 28 µM dithiothreitol and was kept at 4°C. PKA was introduced into
the perfusion system once a seal had formed.
ATP (disodium salt), 8-bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP; sodium salt), PKA, 4-AP, and iberiotoxin were all obtained from Sigma (St. Louis, MO).
Statistics.
Because the control and experimental values were both obtained for the
same patches, data were analyzed by Student's paired t-test and the
2 test. The results are
expressed as means ± SE. The threshold of significance was
P < 0.05.
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RESULTS |
Activity of the cell-attached patches.
Table 1 summarizes the incidences of the
various ion channels. The properties of each channel type are described
below. Nonselective cation channels (CAN channels) were silent in 90%
of the cell-attached patches, but they were found in one-half of the
excised patches and have been described in previous studies (31, 32).
Many cell-attached patches contained no spontaneously active ion
channels, but they were significantly less frequent when 100 µM
acetylcholine was in the pipette medium (38% silent cell-attached
patches with acetylcholine vs. 64% without acetylcholine,
P < 0.02). The frequency of
high-conductance K+ channels was
not significantly modified by the presence of acetylcholine (16% of
cell-attached patches without acetylcholine vs. 12% with acetylcholine). On the contrary, significantly more low-conductance K+ channels were detected with
acetylcholine inside the pipette (25% without acetylcholine vs. 56%
with acetylcholine, P < 0.004). This
property was used to study the low-conductance
K+ channel. In contrast, the
superfusion of acetylcholine outside pipettes containing a control
solution without acetylcholine did not modify the activity of
spontaneously active K+ channels
(n = 4) and did not stimulate
K+ channels in inactive
cell-attached patches (n = 4).
Superfusion with 100 µM adenosine in the bath could activate
low-conductance K+ channels in
cell-attached patches when the pipettes did not contain acetylcholine.
High-conductance
K+ channels.
Figure 1C
shows the typical activity of the high-conductance channels in the
cell-attached and inside-out configuration. These channels were found
in 22 of 102 patches (21%) and usually had a low resting
Po in the
cell-attached configuration but could clearly be identified before
excision in a majority of cases (15 of 22 patches, 68%) in negative
potentials and low positive potentials. However, it was difficult to
clearly identify openings in potentials greater than
60 mV,
probably because the flickery activity of the channels could not be
distinguished from the background noise. Their activity was immediately
increased after excision into a bath containing 1 mM
CaCl2 (compare top and bottom
recordings in Fig. 1C). No rundown
was observed even several minutes after excision. These channels had a
high unit conductance (216 ± 4 pS,
n = 5) and a linear
i-V
relationship under symmetrical 145 mM
K+ conditions (Fig.
1B). The
i-V
relationship showed a slight rectification when the
K+ at the cytoplasm surface was
reduced from 145 to 5 mM and could be fitted by a Goldman equation. The
reversal potential of the I-V
relationship was shifted to
46 ± 3 mV (Fig.
1B), indicating a preference for
K+ over
Na+
(PK/PNa = 8). This channel, which is activated by depolarization (see Fig.
1A) and by an increase in internal
Ca2+ (data not shown), had
properties similar to those of the
maxi-K+ channel described by
Ashmore and Meech (2) in the guinea pig OHC. The channel was closed at
all voltages with 10
9 M
CaCl2 in the bath solution
(n = 8) and maximally activated for
positive voltages at 10
5 M
CaCl2
(n = 8, Po = 0.65 ± 0.10, Vm=
60 mV; see Fig. 1A). The sensitivity of high-conductance channels to iberiotoxin, a specific blocker of maxi-K+ channels, was
studied in excised inside-out patches. The tips of patch pipettes were
filled with iberiotoxin-free solution and then back-filled with
solution containing 100 nM iberiotoxin as described by Jackson and
Blair (14). Blockade was complete 1-2 min after the gigaohm seal
was obtained in all patches containing a spontaneous activity of
high-conductance K+ channels after
excision (n = 4, see Fig.
2C). The
sensitivity of high-conductance channels on the internal side of the
membrane to other blockers of K+
channels such as barium, tetraethylammonium (TEA), and quinine was
studied in inside-out patches. Figure
2A shows a typical experiment in which
the effects of barium (1 mM, n = 4)
and TEA (10 mM, n = 3) were studied at
0 mV. Barium almost completely blocked the channel without altering the
unit conductance, whereas the most notable effect of TEA was to reduce
the conductance from 216 ± 4 pS (n = 5) to 166 ± 19 pS (n = 3). Quinine reduced the
Po from 0.60 ± 0.1 (n = 3) to 0.30 ± 0.08 (n = 3) and also caused a rapid flickering.

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Fig. 1.
Properties of high-conductance K+
channel. A: typical inside-out
recordings of high-conductance K+
channel at various membrane voltages
(Vm). Note
sensitivity of channel to voltage and activity of flickering bursts.
CaCl2 concentration on inner side
of membrane was 10 5 M. Note
presence of smaller events with long openings that could correspond to
another K+ channel or
Ca2+-sensitive nonselective cation
(CAN) channels. C and bars to left of
each trace indicate closed-state current.
B: current
(I)-voltage
(V) relationships in cell-attached
and excised inside-out patches under various conditions. Pipette
contained 145 mM KCl and 2 mM EGTA without
CaCl2. Command potential
(Vc) was
identical to Vm
for inside-out patches but represents
Vm Em for
cell-attached patches, where spontaneous membrane potential
(Em) is
unknown. Open triangles indicate mean ± SE current amplitudes of 6 cell-attached patches, and line represents least-squares fit to Goldman
relationship. Intercept with abscissa was extrapolated to 57 mV,
because there were no clear openings attributed to these channels at
positive potentials. Open circles indicate mean ± SE currents of 5 excised inside-out patches, with bath containing 145 mM KCl, and line
is linear regression of points weighted at each voltage by SE. Unit
conductance was 216 ± 4 pS, and reversal potential was near 0 mV.
Solid circles are mean ± SE currents of 6 excised inside-out
patches, with bath containing 140 mM NaCl and 5 mM KCl. Line is
least-squares fit to Goldman relationship. Reversal potential is
shifted to an extrapolated value of 46 mV, close to Nernst
equilibrium potential for K+.
PK/PNa,
relative permeabilities of K+ and
Na+, was 8. Error bars are
indicated when SE exceeded size of symbols.
C: typical recordings of
high-conductance K+ channels in
cell-attached (top trace) and
inside-out configurations (bottom
trace).
Vc is 0 mV, and C
indicates closed level. Note typical behavior with burst of
"flickering" activity with periods of silence. Activity is
increased after excision with bath containing 1 mM
CaCl2.
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Fig. 2.
Effects of K+ channel blockers.
A: single-channel recordings of an
inside-out patch containing one level of high-conductance
K+ channel at 0 mV.
Top trace is control.
Middle trace shows typical effect of
internal 10 mM tetraethylammonium (TEA). Amplitude of current is
reduced from 6 to 3 pA, but open probability
(Po) is
unchanged. Bottom trace shows effect
of 1 mM internal barium.
Po is greatly
reduced. B: single-channel recordings
of an inside-out patch containing low-conductance
K+ channels at 0 mV.
Top trace is control. Note flickering
activity but at a slower rate than in
A. Middle
trace shows effects of 10 mM internal TEA. Amplitude of
current is reduced from 1 to 0.6 pA. Bottom
trace shows effect of barium.
Po is reduced,
but amplitude of events remains unchanged.
C: continuous single-channel recording
of a patch containing one level of high-conductance
K+ channel.
Vc is 0 mV, and C
indicates closed level. Arrow indicates excision from cell-attached to
inside-out configuration. Tips of pipettes were filled with
iberiotoxin-free solution and then back-filled with a solution
containing 100 nM iberiotoxin.
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Low-conductance
K+ channels.
Low-conductance K+ channels were
more frequently detected when there was acetylcholine in the pipette,
and they opened spontaneously in the cell-attached configuration. This
activity was usually bursts of openings separated by periods of silence
lasting from several milliseconds to seconds (see Fig.
3, A and
C). Excision was followed by a
gradual rundown of the low-conductance
K+ channels in most patches (34 of
44, 77%; see Fig. 3C).
Acetylcholine in the pipette or 1 mM ATP
(n = 3) in the bath did not prevent this phenomenon. Rundown was not observed for the high-conductance K+ channels and for the
nonselective cation channels. The
i-V
relationship in the cell-attached configuration was curvilinear, with a
slight inward rectification probably due to the low resting potential of isolated OHC and their low internal
K+ concentration. However, the
channel in excised patches had a slight outward rectification in
symmetrical K+ concentrations (see
Fig. 3B). The slope conductance was
greater at positive
Vm (21.5 ± 2.4 pS, n = 6) than at negative
Vm (10.3 ± 1.7 pS, n = 6). The
i-V
relationship showed strong rectification with a NaCl-rich solution in
the bath and the reversal potential could be extrapolated to
67.4 mV, a value very near the calculated equilibrium potential
for K+ (
85 mV). Although
this
i-V
relationship did not fit a Goldman relationship very well, it was
assumed to be valid near the reversal potential. The
PK/PNa
was 28. Replacing the bath NaCl with RbCl showed no clearly
identifiable openings in positive potentials. These results suggest a
low permeability or blockade of the channel by
Rb+ (data not shown,
n = 3), as described in
maxi-K+ channels of vestibular
dark cells (30). The channel was blocked by internal barium (1 mM,
n = 3) and TEA (10 mM,
n = 4), as was the high-conductance
channel. TEA also caused a "flickery" blockade of the channel
with a smaller current amplitude (16 ± 8% of control) and
decreased Po (27 ± 6% of the control values, see Fig.
2B). Barium blocked the channel
almost completely by decreasing the Po to 11 ± 6% of the control value without altering the current amplitude. This
type of channel was also blocked by 4-AP, which inhibits the
voltage-gated delayed rectifier K+
channel (see Fig.
4A). The
conductance of the channel remained unchanged, but the
Po was reduced
with 10
4 M 4-AP
(Po = 72 ± 6% of the control value,
n = 4, P < 0.02) and was almost completely
blocked by 10
3 M 4-AP
(Po = 17 ± 4% of the control value, n = 4, P = 0.001).

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Fig. 3.
Single-channel recordings and conductive properties of low-conductance
K+ channel.
A: typical inside-out recordings of
low-conductance K+ channel at
various membrane voltages
(Vm). Note low
voltage sensitivity of channel and bursting activity.
CaCl2 concentration on inner side
of membrane was 10 9 M. C
and bars to left of each trace
indicate closed state current. B:
I-V
relationships in cell-attached and excised inside-out patches under
various conditions. Pipette contained 145 mM KCl and 2 mM EGTA without
CaCl2.
Vc was identical
to Vm for
inside-out patches but represents
Vm Em for
cell-attached patches, where spontaneous membrane potential
Em is
unknown. Open triangles indicate mean ± SE
currents of 6 cell-attached patches. Intercept with abscissa was
64 mV. Open circles indicate mean ± SE currents of 6 excised
inside-out patches, with bath containing 145 mM KCl. Slope conductance
was significantly greater in positive
Vm potentials
(21.5 ± 2.4 pS, n = 6) than in
negative Vm
potentials (10.3 ± 1.7 pS, n = 6),
and reversal potential was near 0 mV. Solid circles are mean ± SE
currents of 6 excised inside-out patches, with bath containing 140 mM
NaCl and 5 mM KCl. Reversal potential is shifted to 67.4 mV,
close to Nernst equilibrium potential for
K+.
PK/PNa,
relative permeabilities of K+ and
Na+, was 28. Error bars are
indicated when SE exceeded size of symbols. Lines are least-squares fit
to a third-order polynomial relationship.
C: typical recordings in cell-attached
(top trace) and inside-out
configurations (top and
bottom trace) of low-conductance
K+ channels.
Vc is 0 mV, and C
indicates closed level. There is a gradual rundown after excision in
bath containing 10 9 M
CaCl2. Bottom
trace shows activity of low-conductance channels with a
shorter time scale.
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Fig. 4.
Sensitivity of low-conductance K+
channel to 4-aminopyridine (4-AP). A:
typical recordings of an inside-out patch containing 3 levels of
low-conductance channel and showing blockade by 4-AP at various
concentrations. Bottom trace shows
recovery from blockade. B: dose-effect
relationship of inhibition of low-conductance
K+ channel by 4-AP. Histograms are
mean Po for 4 patches in control solution,
10 4 M 4-AP, and
10 3 M 4-AP.
* P < 0.02 when compared with
control. ** P = 0.001 when
compared with control (Student's paired
t-test).
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Voltage dependence and kinetics of
K+ channels.
Both types of K+ channel were
voltage dependent. Figure
5B shows
their voltage and
Po. The
Po did not exceed
0.8 for the high-conductance K+
channel and 0.6 for the low-conductance
K+ channel, even when the patches
were strongly depolarized, probably because of the flickering activity
of the channels. The half-maximal activation potential was
43 mV
for the high-conductance K+
channel (n = 6) with 10 µM internal
Ca2+, and
15 mV
(n = 7) for the low-conductance
channel, indicating that the channels were sensitive over different
ranges of voltage. We investigated the mechanism underlying this
dependence by analyzing recordings containing only one level of
channel. The kinetics of the two types of channels were best described
by two time constants for the open state and two time constants for the
closed state (see Figs. 6 and
7). The results are summarized in Tables
2 and 3. The voltage affected the long closed
time constant in both channels, indicating that depolarization
increased the Po
by reducing the interval between bursts of activity. However,
depolarization increased both the open time constants only for
high-conductance channels, indicating a modification of intraburst
activity (Fig. 6). Analysis of the blockade by TEA revealed that the
time constants were not affected. Blockade of the low-conductance
K+ channel was of a different
type, as it decreased the open time constant without altering the
closed time constants.

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Fig. 5.
Voltage dependence of two types of
K+ channels.
A: representative single-channel
recordings obtained from an inside-out patch containing both high- and
low-conductance K+ channel at
various voltages. C and bars to left
of each trace indicate closed state current. Internal concentration of
CaCl2 was
10 5 M. Note differences in
voltage dependence of channels. High-conductance channel has a clear
activity only at positive membrane potential
(Vm).
Po of
low-conductance K+ channels is
reduced at negative potentials, but openings are clearly identified.
B: relationship between
Po and
Vm. Open circles
are mean ± SE
Po of 6 excised
inside-out patches containing high-conductance
K+ channels and exposed to
10 5 M
CaCl2 in bath, and solid circles
indicate mean ± SE
Po of 7 excised
inside-out patches containing low-conductance
K+ channels exposed to
10 9 M
CaCl2. Data points were fitted to
a modified Boltzman equation using Marcquardt method of least squares
(see METHODS for details).
Half-maximal voltages were 43 mV (high conductance) and
15 mV (low conductance).
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Fig. 6.
Steady-state kinetic properties of high-conductance
K+ channel.
A: histograms of closed
(left) and open
(right) times of an inside-out patch
containing one level of high-conductance channel at a membrane
potential of 0 mV. Recording lasted 60 s; total number of events was
1,811; and Po was
0.06. Pipette contained 145 mM KCl and 2 mM EGTA, and bath contained
140 mM NaCl, 5 mM KCl, and
10 5 M
CaCl2. Data were low-pass filtered
at 2 kHz and digitized at 5 kHz, and idealized recordings were
constructed. Curves were fitted to sum of 2 exponentials to dwell-time
data by a least-squares method. Events shorter than 0.5 ms were
excluded from fit. Time constants are given on each panel:
1 is fast time constant, and
2 is slow time constant. Line
corresponds to fit of data with two exponentials.
Inset in left
panel shows analysis of long closures (longer than 10 ms) that were assumed to correspond to interval between bursts.
Distribution of closures fits well with a single exponential, and
number of closures of 10-400 ms was 473. B: closed-time constants
(left) and open-time constants
(right) are shown as a function of
membrane potential. Plot shows mean time constants obtained from fits
of 2-4 patches like those illustrated in
A (see Table 3 for values). Open
circles represent long time constants, and solid triangles represent
short time constants.
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Fig. 7.
Steady-state kinetic properties of low-conductance
K+ channel.
A: histograms of closed
(left) and open
(right) times of an inside-out patch
containing one level of low-conductance channel at a membrane potential
of 0 mV. Recording lasted 60 s, total number of events was 13,660, and
Po was 0.43. Pipette contained 145 mM KCl and 2 mM EGTA, and bath contained 140 mM
NaCl, 5 mM KCl, and 10 9 M
CaCl2. Data were low-pass filtered
at 1 kHz and digitized at 3 kHz, and idealized recordings were
constructed. Curves were fitted to sum of 2 exponentials to dwell-time
data by a least-squares method. Events shorter than 1 ms were excluded
from fit. Time constants are given on each panel:
1 is fast time constant, and
2 is slow time constant. Line
corresponds to fit of data with two exponentials.
B: closed-time constants
(left) and open-time constants
(right) are shown as a function of
membrane potential. Mean time constants obtained from fits of 3-5
patches like those illustrated in A
are plotted (see Table 2 for values). Open circles represent long time
constants, and solid triangles represent short time constants.
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Table 2.
Open probability and time constants for low-conductance potassium
channel in response to depolarization, blockers, and activators
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Table 3.
Open probability and time constants for high-conductance potassium
channel in response to depolarization, blockers, and activators
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|
We also investigated the properties of the low-conductance
K+ channel by ensemble-averaged
2-s sweeps in response to pulses applied to
40 mV from a holding
potential of
60 mV. The associated mean current produced by 20 steps is shown in Fig. 8. The current showed a very slow inactivation with a time constant of ~800 ms.

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Fig. 8.
Low-conductance K+ current
measured by cell-attached averaged single-channel recording. Pipette
solution contained 145 mM KCl, and bath solution contained 140 mM NaCl
and 5 mM KCl. Leak and capacitive currents were subtracted from all
sweeps. Data were recorded with low-pass filtering at 2 kHz and
digitized at 5 kHz. Cell-attached patch was clamped to a potential of
40 mV positive to holding potential ( 60 mV). Five
representative sample traces are shown below voltage pulse.
Ensemble-averaged current obtained from 20 consecutive sweeps is shown
below 5 sample traces. Bottom trace
shows a single sweep before subtraction of leak and capacitive
currents.
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|
Effects of external purines.
In a previous study (32), we demonstrated that superfusion with
purinergic agonists modulated the activity of a
Ca2+-sensitive nonselective cation
channel (CAN) in the basolateral membrane of OHC. ATP increased the
activity of CAN, probably by increasing intracellular
Ca2+, and adenosine reduced the
activity of CAN, probably by increasing intracellular cyclic AMP. We
therefore investigated the effects of external ATP and adenosine on
K+ channel activities using the
following protocol. After obtaining a seal, the cells were superfused
with control saline solution (140 mM NaCl and 4.8 mM KCl) for 1-2
min to check for spontaneous or stretch-induced activation, and then
challenged with ATP or adenosine. The pipettes were filled with a high
K+ solution and currents were
recorded at the resting membrane potential (clamp potential = 0 mV)
near the reversal potential for CAN to minimize their amplitude. ATP
had no effect on maxi-K+
(n = 9) or low-conductance
K+ channels
(n = 3). ATP did not alter the
activity of spontaneous opening
maxi-K+ channels
(n = 4) or stimulate
maxi-K+ channels in inactive
cell-attached patches having
maxi-K+ channel activity after
excision (n = 5). Adenosine also did
not alter the activity of maxi-K+
channels (n = 5). Purines had no
effect on the maxi-K+ channel as
evaluated by the
Po or the kinetic
analysis (data not shown, n = 5).
However, in the study where we reported the effects of adenosine on CAN
(32), some patches frequently showed activation of small flickering
channels, possibly the small K+
channels, but the ionic selectivity of these channels was not investigated because the pipette contained a low
K+ concentration (4.8 mM). We
therefore reinvestigated the effects of external adenosine in
cell-attached patches; the pipette contained a control 145 mM KCl
solution without acetylcholine to reduce the spontaneous activity of
the low-conductance K+ channels.
Eight of thirteen patches responded with a significant increase in
NPo from 0.36 ± 0.19 to 0.54 ± 0.19 (P < 0.005) with a delay of 51 ± 9 s (Fig.
9). This effect was reversed by washing out
the activator in only four of eight cases. Kinetic analysis of the
channel activity before and during the superfusion of adenosine revealed that the open time constants were not modified but that the
slow closed time constant was increased.

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Fig. 9.
Reversible cell-attached activation of low-conductance
K+ channels by adenosine.
A: continuous trace of low-conductance
K+ channel in cell-attached
configuration at a
Vc of 30
mV before (open horizontal bar), during (solid horizontal bar), and
after (open horizontal bar) superfusion of 100 µM adenosine. Pipette
contained 145 mM KCl, and bath contained 140 mM NaCl and 5 mM KCl.
Delay before activation was 50 s. C indicates closed level. Note
multiple peaks that probably correspond to short openings of
high-conductance K+ channels and seem not to be influenced
by adenosine. B: histograms showing
time course of channel activity,
NPo, where
N is total number of channels and
Po is open-state
probability, before (open horizontal bar, 30 s), during exposure to 100 µM adenosine (solid horizontal bar, 60 s), and after washout of
adenosine. C: bar graph showing
activity of low-conductance K+
channels in 8 inside-out patches stimulated with 100 µM adenosine.
NPo was increased
from 0.36 ± 0.19 to 0.54 ± 0.19 (P < 0.005 by paired Student's
t-test).
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|
Activation by 8-BrcAMP and PKA.
Because the inhibition of CAN by adenosine is correlated with the
effect of cAMP and PKA, we attempted to determine whether this
possibility also held for the K+
channel. Of the 10 patches tested in the cell-attached configuration with 0.5 mM 8-BrcAMP, 5 clearly responded with a mean delay of 61 ± 15 s and increased their resting
NPo significantly
from 0.61 ± 0.27 to 0.92 ± 0.32 (P < 0.02). Eight of eleven
inside-out excised patches increased their resting
NPo significantly
from 0.54 ± 0.04 to 0.89 ± 0.02 (P < 0.001) in response to PKA with
a mean delay of 80 s (see Fig. 10).
Kinetic analysis of four patches containing only one level of
low-conductance K+ channel showed
that only the slow closed time constant was altered. This effect was
not significant (P = 0.10),
probably because the sample was too small. Nevertheless, these results
suggest that the activity of the channel is altered by adenosine and
PKA via a common mechanism.

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Fig. 10.
Activation of low-conductance K+
channels in inside-out configuration by catalytic subunit of protein
kinase A (PKA; 20 U/ml). A: sample
traces of low-conductance K+
channel recorded in inside-out configuration before
(top trace) and after superfusion of
PKA (bottom trace) at a
Vc of 0 mV.
Pipette contained 145 mM KCl, and bath contained 140 mM NaCl and 5 mM
KCl. Delay before activation was 1 min. C indicates closed level.
B: diagram indicates change in mean
channel activity
NPo of 8 patches
with time before (open horizontal bar, 30 s) and during exposure to PKA
(solid horizontal bar). Open circles represent means of overall
NPo values before
and during activation.
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|
 |
DISCUSSION |
We have shown that there are two voltage-sensitive
K+ channels in the basolateral
membrane of OHC from the guinea pig cochlea. These channels are readily
distinguished from each other by their conductance; one is ~200 pS
and the other 20 pS. The high-conductance channel is
Ca2+ sensitive, blocked by
iberiotoxin, and clearly belongs to the family of
maxi-K+ channels previously
described in these cells. TEA reduced the conductance of the channel,
suggesting a fast block, as described in other cells of the inner ear
(30). The low-conductance channel is
Ca2+ insensitive and is blocked by
barium, TEA, and 4-AP. There seem to be more low-conductance channels
than maxi-K+ channels, but the
slow inactivation in cell-attached patches and the existence of a
rundown phenomenon after excision makes them more difficult to study,
and probably explains why they have not been reported previously in
single-channel recordings of OHC.
Activity and regulation of the high-conductance
K+ channel.
The high-conductance channel described here is very like the large
Ca2+-activated
K+ channel reported by Ashmore and
Meech in 1986 (2) and the C-type
K+ channel found by Gitter et al.
in 1992 (9). The channel opens for short periods in situ and was
clearly identified in most cases, probably because it is very sensitive
to Ca2+, and the resting
intracellular Ca2+ concentration
of isolated OHC is above the threshold of activation of the channel.
The Po was
greatly increased after excision in a
Ca2+-rich bath, indicating its
Ca2+ sensitivity. We tested only
10
9,
10
5, and
10
3 M
CaCl2 concentrations, but the
threshold of activation has not been precisely measured. Ashmore and
Meech (2) found a low threshold near
10
8 M in the guinea pig
cochlea, Art et al. (1) examined turtle hair cells, and Sugihara (29)
studied goldfish hair cells; they found that the
Po/Vm
relationship depended greatly on the intracellular Ca2+ concentration. We find that
the voltage dependence in inside-out patches with 10 µM
CaCl2 bathing the inner side of
the membrane is in the same range, which indicates a low but
significant Po when the membrane potential has low negative values. Kinetic analysis demonstrates that the activity of these channels is made up of bursts
and is well described with two open and two closed state constants.
Depolarization decreases the long closed time constant, thus reducing
the time between bursts, and increases the open time constants,
indicating stabilization of the open state. Purines do not alter the
Po or the
intraburst behavior of the channels. These findings are compatible with
the fact that voltage is the main regulator of these channels when
intracellular Ca2+ level is kept constant.
Properties and regulation of the low-conductance
K+ channel.
The precise nature of the low-conductance channel is not yet clear.
However, the voltage dependence, the small conductance of 10-20
pS, its insensitivity to internal
Ca2+, the outward rectification,
the bursts of activity, the inhibition by 4-AP, and the slow
inactivation in averaged single traces all point to it being a delayed
rectifier K+ channel. Lin et al.
(18) and Nenov et al. (23) recently described a similar type of
K+ current in whole cell
recordings of guinea pig OHC and indicated that it could play an
important part in generating the
Ca2+-insensitive outwardly
rectifying K+ current. This
current is blocked by submillimolar concentrations of 4-AP, is
inactivated slowly, and could well be the low-conductance K+ channel described here.
We found that the low-conductance
K+ channel is regulated by several
factors. The channel is significantly more frequent in patches obtained
with an acetylcholine-filled pipette, suggesting local interaction
between the mediator and the channel. In addition, external adenosine
activates the channel when superfused outside the pipette. This implies
activation by an internal second messenger, probably cAMP. This is
reinforced by the fact that PKA significantly increases channel
activity. Kinetic analysis points to adenosine and PKA acting via a
common mechanism, as both decrease the slow closed time constant.
Putative role of these channels.
The main function of basolateral
K+ channels is to maintain the
cell membrane potential under resting conditions and to cause hyperpolarization in response to activators, and thus reduce the excitability of the OHC. Most of the previous single-channel recordings of K+ channels in the basolateral
membrane of guinea pig OHC reported Ca2+-sensitive
K+ channels, with the majority
being the high-conductance maxi-K+
or BKCa channels. The effects of
K+ channel blockers, like barium,
TEA, and cesium, suggested that Ca2+-activated channels carry most
of the voltage-dependent outwardly rectifying
K+ current. The voltage
sensitivities of the high-conductance and low-conductance
K+ channels in our study reveal
that the low-conductance channel has a higher
Po than the
high-conductance channel when the membrane potential of OHC is more
negative than
30 mV and the intracellular Ca2+ concentration is low. It
could thus play an important role in resting conditions. Recent in vivo
studies (33) showed that the superfusion of 5 mM 4-AP in the perilymph
of guinea pig cochleas (on the basolateral membrane of hair cells)
increased the summating potential at test frequencies below 4 kHz,
where the major contribution is thought to come from the OHC. These
results therefore suggest that 4-AP-sensitive
K+ channels like our
low-conductance K+ channels are
important under physiological conditions for clamping the membrane
potential at more negative values.
Acetylcholine is thought to be the main neurotransmitter of the medial
efferent system that sends many fibers directly to the base of the OHC
(for a review, see Ref. 8) and may be the agent of neural control of
the OHC by acting on basolateral
K+ channels. Recent studies using
whole cell recordings in guinea pig OHC indicate that acetylcholine
triggers a brief cationic nonselective current that allows
Ca2+ to permeate and thus
immediately activate the neighboring
Ca2+-sensitive
K+ channels, probably
low-conductance ones that typically adapt rapidly (4, 12, 15, 21, 22).
Although recorded at the base of the OHC, our low-conductance
K+ channels are clearly distinct
from these acetylcholine-related K+ channels, because they are
insensitive to internal Ca2+ and
are still activated after a few minutes of contact between the pipette
containing acetylcholine and the membrane during the formation of the
seal. Our low-conductance K+
channels could thus produce a long-lasting hyperpolarization of the
OHC. Slow inhibitory effects of efferent stimulations with a latency of
10-50 s have been observed recently in the cochlea (5, 28). The
authors suggest that these effects are caused by a more delayed
intracellular mechanism, probably involving second messengers or
phosphorylation. Acetylcholine was found to increase the activity of
low-conductance K+ channels when
present in the pipette, but not when added to the bath while the
pipette contained control solution. This could indicate a mechanism
distinct from the activation by adenosine and PKA, such as G proteins
(15). Our low-conductance K+
channels, which are activated by acetylcholine and regulated by cAMP
and PKA, could take part in this slow inhibition of OHC.
Purinergic receptors have been found in the apical and basolateral
membranes of OHC, although their precise function is unclear. The organ
of Corti itself could participate in the release of ATP (34). We
previously studied the regulation by purines of a CAN that could
depolarize the cell and thus increase its excitability. These channels
were activated by ATP and inhibited by adenosine, its precursor and
degradation product. The two types of basolateral K+ channels described here are
activated by depolarization and/or adenosine. They could cooperate by
balancing the effects of the nonselective cationic channels and so
prevent prolonged depolarizations and injury to the cells.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Tran Ba
Huy, Laboratoire de Neurobiologie des Systèmes Sensori-moteurs,
CNRS, UPRESA 7060, Faculté de Médecine Lariboisière,
Paris Cedex 18, France. (E-mail:
thierry.van-den-abbeele{at}rdb.ap-hop-paris.fr).
Received 22 June 1998; accepted in final form 9 July 1999.
 |
REFERENCES |
1.
Art, J. J.,
Y. C. Wu,
and
R. Fettiplace.
The calcium-activated potassium channels of turtle hair cells.
J. Gen. Physiol.
105:
49-72,
1995[Abstract].
2.
Ashmore, J.,
and
R. W. Meech.
Ionic basis of membrane potential in guinea pig outer hair cells.
Nature
322:
368-371,
1986[Medline].
3.
Barry, P. H.,
and
J. W. Lynch.
Liquid junction potentials and small cell effects in patch-clamp analysis.
J. Membr. Biol.
121:
101-117,
1991[Medline].
4.
Blanchet, C.,
C. Erostegui,
M. Sugasawa,
and
D. Dulon.
Acetylcholine-induced potassium current of guinea pig outer hair cells: its dependencde on a calcium influx through nicotinic-like receptors.
J. Neurosci.
16:
2574-2584,
1996[Abstract].
5.
Da Costa, D. L.,
A. Chibois,
J. P. Erre,
C. Blanchet,
R. C. de Sauvage,
and
J. M. Aran.
Fast, slow, and steady-state effects of contralateral acoustic activation of the medial olivocochlear efferent system in awake guinea pigs: action of gentamicin.
J. Neurophysiol.
78:
1826-1836,
1997[Abstract/Free Full Text].
6.
Elgoyhen, A. B.,
D. S. Johnson,
J. Boulter,
D. E. Vetter,
and
S. Heinemann.
Alpha9: an acetylcholine receptor with novel parmacological properties expressed in rat cochlear hair cells.
Cell
79:
705-715,
1994[Medline].
7.
Erostegui, C.,
A. P. Nenov,
C. H. Norris,
and
R. P. Bobbin.
Acetylcholine activates a K+ conductance permeable to Cs+ in guinea pig outer hair cells.
Hear. Res.
81:
119-129,
1994[Medline].
8.
Eybalin, M.
Neurotransmitters and neuromodulators of the mammalian cochlea.
Physiol. Rev.
73:
309-373,
1993[Free Full Text].
9.
Gitter, A. H.,
E. Frömter,
and
H. P. Zenner.
C-type potassium channels in the lateral cell membrane of guinea pig outer hair cells.
Hear. Res.
60:
13-19,
1992[Medline].
10.
Glowatzki, E.,
K. Wild,
U. Brändle,
G. Fakler,
B. Fakler,
H. P. Zenner,
and
J. P. Ruppersberg.
Cell-specific expression of the
9 n-Ach receptor subunit in auditory hair cells revealed by single-cell RT-PCR.
Proc. R. Soc. Lond. B Biol. Sci.
262:
141-147,
1995[Medline].
11.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
12.
Housley, G. D.,
and
J. F. Ashmore.
Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea.
Proc. R. Soc. Lond. B Biol. Sci.
244:
161-167,
1991[Medline].
13.
Housley, G. D.,
and
J. F. Ashmore.
Ionic currents of outer hair cells isolated from the guinea pig cochlea.
J. Physiol. (Lond.)
448:
73-98,
1992[Abstract].
14.
Jackson, W. F.,
and
K. L. Blair.
Characterization and function of Ca2+-activated K+ channels in arteriolar muscle cells.
Am. J. Physiol.
274 (Heart Circ. Physiol. 43):
H27-H34,
1998[Abstract/Free Full Text].
15.
Kakehata, S.,
T. Nagakawa,
T. Tasakasa,
and
N. Akaike.
Cellular mechanisms of acetylcholine-induced response in dissociated outer hair cells of guinea pig cochlea.
J. Physiol. (Lond.)
463:
227-244,
1993[Abstract].
16.
Levitan, I. B.
Modulation of ion channels by protein phosphorylation and dephosphorylation.
Annu. Rev. Physiol.
56:
193-212,
1994[Medline].
17.
Liberman, M. C.,
L. W. Dodds,
and
S. Pierce.
Afferent and efferent innervation of the cat cochlea: quantitative analysis with light and electron microscopy.
J. Comp. Neurophysiol.
301:
443-460,
1990.
18.
Lin, X.,
R. I. Hume,
and
A. L. Nuttal.
Dihydropyridines and verapamil inhibit voltage-dependent K+ current in isolated outer hair cells of the guinea pig.
Hear. Res.
88:
36-46,
1995[Medline].
19.
Mammano, F.,
and
J. F. Ashmore.
Differential expression of outer hair cell potassium currents in the isolated cochlea of the guinea pig.
J. Physiol. (Lond.)
496:
639-646,
1996[Abstract].
20.
Miller, D. J.,
and
G. L. Smith.
EGTA purity and the buffering of calcium ions in physiological solutions.
Am. J. Physiol.
246 (Cell Physiol. 15):
C160-C166,
1984[Abstract/Free Full Text].
21.
Nenov, A. P.,
C. Norris,
and
R. P. Bobbin.
Acetylcholine response in guinea pig outer hair cells. I. Properties of the response.
Hear. Res.
101:
132-148,
1996[Medline].
22.
Nenov, A. P.,
C. Norris,
and
R. P. Bobbin.
Acetylcholine response in guinea pig outer hair cells. II. Activation of a small conductance Ca2+-activated K+ channel.
Hear. Res.
101:
149-172,
1996[Medline].
23.
Nenov, A. P.,
C. Norris,
and
R. P. Bobbin.
Outwardly rectifying currents in guinea pig outer hair cells.
Hear. Res.
105:
146-158,
1997[Medline].
24.
Paulais, M.,
and
J. Teulon.
A cation channel in the thick ascending limb of Henle's loop of the mouse kidney: inhibition by adenine nucleotides.
J. Physiol. (Lond.)
413:
315-327,
1989[Abstract].
25.
Sadoshima, J. I.,
N. Akaike,
H. Kanaide,
and
M. Nakamura.
cAMP modulates Ca2+-activated K+ channels in cultured smooth muscle cells of rat aortas.
Am. J. Physiol.
255 (Heart Circ. Physiol. 24):
H754-H759,
1988[Abstract/Free Full Text].
26.
Santos-Sacchi, J.,
and
J. P. Dilger.
Whole cell currents and mechanical responses of isolated outer hair cells.
Hear. Res.
35:
143-150,
1988[Medline].
27.
Spoendlin, H.
Innervation patterns of the organ of Corti of the cat.
Acta Otolaryngol.
67:
239-254,
1969[Medline].
28.
Sridhar, T. S.,
M. C. Liberman,
M. C. Brown,
and
W. F. Sewell.
A novel cholinergic "slow effect" of efferent stimulation, cochlear potentials in the guinea pig.
J. Neurosci.
15:
3667-3678,
1995[Abstract].
29.
Sugihara, I.
Calcium-activated potassium channels in goldfish hair cells.
J. Physiol. (Lond.)
476:
373-390,
1994[Abstract].
30.
Takeuchi, S.,
D. C. Marcus,
and
P. Wangemann.
Maxi K+ channel in apical membrane of vestibular dark cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C1430-C1436,
1992[Abstract/Free Full Text].
31.
Van Den Abbeele, T.,
P. Tran Ba Huy,
and
J. Teulon.
A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea pig cochlea.
Pflügers Arch.
417:
56-63,
1994.
32.
Van Den Abbeele, T.,
P. Tran Ba Huy,
and
J. Teulon.
Modulation by purines of a nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea pig cochlea.
J. Physiol. (Lond.)
494:
77-89,
1996[Abstract].
33.
Van Emst, M. G.,
S. F. L. Klis,
and
G. F. Smoorenburg.
4-Aminopyridine effects on summating potentials in the guinea pig.
Hear. Res.
102:
70-80,
1996[Medline].
34.
Wangemann, A. P.
Calcium-dependent release of ATP from the organ of Corti measured with a luciferin-luciferase bioluminescence assay.
Auditory Neurosci.
2:
187-192,
1996.
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