Voltage-dependent outward K+
current in intermediate cell of stria vascularis of gerbil
cochlea
Shunji
Takeuchi and
Motonori
Ando
Department of Physiology, Kochi Medical School, Nankoku 783-8505, Japan
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ABSTRACT |
A voltage-dependent outward
K+
(KV) current in the intermediate
cell (melanocyte) of the cochlear stria vascularis was studied using
the whole cell patch-clamp technique. The
KV current had an activation
threshold voltage of approximately
80 mV, and 50% activation
was observed at
42.6 mV. The time courses of activation and
inactivation were well fitted by two exponential functions: the time
constants at 0 mV were 7.9 and 58.8 ms for activation and 0.6 and 4.3 s
for inactivation. The half-maximal activation time was 13.8 ms at 0 mV.
Inactivation of the current was incomplete even after a prolonged
depolarization of 10 s. This current was independent of intracellular
Ca2+. Quinine, verapamil,
Ba2+, and tetraethylammonium
inhibited the current in a dose-dependent manner, but 4-aminopyridine
was ineffective at 50 mM. We conclude that the
KV conductance in the intermediate
cell may stabilize the membrane potential, which is thought to be
closely related to the endocochlear potential, and may provide an
additional route for K+ secretion
into the intercellular space.
patch clamp; melanocyte; endocochlear potential
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INTRODUCTION |
IT HAS BEEN GENERALLY ACCEPTED that the stria
vascularis in the cochlea is responsible for the production of the
positive endocochlear potential (EP) and the
K+-rich endolymph. Both the EP and
the high K+ concentration in the
endolymph are essential for sound transduction by hair cells. The
mechanoelectrical transducer channels in the cilia of hair cells make
contact with the endolymph, and K+
flows into hair cells when the transducer channels open. The driving
force for the K+ flux through the
transducer channels is the electrical gradient across the ciliary
membrane produced by the sum of the EP (+80 to +90 mV) and
the resting membrane potential of hair cells (
40 to
50 mV
for inner hair cells) (7). Accordingly, the cochlear microphonic
potential decreases when the EP is suppressed (21).
The stria vascularis consists of several types of cells: marginal
cells, intermediate cells, basal cells, capillary endothelial cells,
and pericytes. Figure 1 shows major
ionic pathways in cells constituting the stria vascularis and the
underlying spiral ligament. Among several cell types, intermediate
cells are melanocytes, which migrate from the neural crest during
ontogeny (12) to become located between the epithelial marginal cell
layer and the mesodermal basal cell layer. Intermediate cells play an
essential role in the development and/or in the physiological function
of the stria vascularis, because a congenital deficiency in the
intermediate cell causes low EP (4, 34) and an increase in the
threshold of sound pressure levels to elicit compound action potential
responses (4).

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Fig. 1.
Schematic drawing of the stria vascularis and spiral ligament based on
review by Wangemann and Schacht (43) and our recent work (36, 37).
Major ionic pathways are shown. minK, slowly activating K+
channel; KIR, inward rectifier K+ channel;
KV, depolarization-activated
K+ channel reported in this study;
b, basal cell; e, endothelial cell and pericyte; f, fibrocyte; i,
intermediate cell; m, marginal cell; tj, tight junction. Subcellular
localization of the K+ channel in
the basal cell is speculative, although a
Ca2+-activated large-conductance
K+ channel has been reported
(39).
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K+ channels in the intermediate
cell and/or the basal cell are thought to play an important role in the
generation of EP and in the recycling of
K+ from the perilymph to the
endolymph (30, 32, 43). We have succeeded in obtaining dissociated
intermediate cells and we reported an inwardly rectifying
K+
(KIR) current in the
intermediate cell (37). The similarity between the drug sensitivity of
the KIR current and that of the EP
suggests a direct contribution of the
KIR conductance to the generation
of the EP (37, 40). In addition to the
KIR current, an outward current in
the intermediate cells was observed when the
KIR current was inhibited by
Ba2+ (37). However, the nature of
the outward current has not yet been investigated. The aim of this
study was to characterize the voltage-dependent outward
K+
(KV) current and to consider its
relevance to the physiological function of the intermediate cell.
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METHODS |
Cell preparation and electrophysiological recordings were performed in
essentially the same way as described previously (37). The care and the
use of animals were approved by the Kochi Medical School Animal Care
and Use Committee. Cochleae of gerbils were obtained under anesthesia
with pentobarbital sodium (50 mg/kg ip). Tissue strips of the stria
vascularis were incubated for 30 min at 24-26°C in the control
solution containing 0.2% trypsin and kept for up to 4 h in the cold
(4°C) control solution until use. These strips were dissected with
fine needles under visual control. Single intermediate cells that
displayed the characteristic morphology (i.e., densely packed pigment
in the cell and dendritelike projections) were selected and separated
from other dissociated cells.
The whole cell configuration of the patch-clamp technique was employed.
Pipette resistance was 2.2-3.6 M
when filled with the pipette
solution. Recordings were made with an amplifier (3900A with 3911A;
Dagan, Minneapolis, MN). Current signals were filtered at 5 kHz through
a four-pole Bessel low-pass filter, digitized at frequencies of
0.5-5 kHz, and stored on the hard disk of a computer. Voltage
command generation, data storage, and analyses were performed using
pCLAMP (version 6.0.3; Axon, Foster City, CA). Membrane capacitance,
estimated using the circuitry of the amplifier, was 23.5 ± 0.6 pF/cell (n = 157). Series resistance (4-15 M
) was compensated as much as possible (by 50-90%)
within a range that did not provoke oscillations of the current,
whereas voltage errors might be significantly large when currents were large. Membrane potentials were corrected for voltage errors derived from uncompensated series resistance (current × uncompensated resistance) when activation curves were made [see Figs. 4
(C and D) and
8D]. Liquid junction potentials
were measured against a flowing 3 M KCl electrode and corrected. Leak
conductance was estimated from the slope of current-voltage relations
of steady-state currents between
110 and
90 mV in the
presence of 0.5 mM Ba2+ (see
RESULTS) and subtracted linearly unless indicated
otherwise, since current-voltage relations of residual currents at
membrane potentials below
90 mV could be regarded as linear.
Experiments were performed at room temperature (24-26°C). Data
were expressed as means ± SE, where
n is the number of cells.
The pipette solution contained (in mM) 110 KCl, 15 potassium aspartate,
10 KOH, 5 EGTA, 6 HEPES, 6 Tris, 1.1 MgCl2, and 2 MgATP (pH, 7.2). The
control bath solution contained (in mM) 110 NaCl, 40 sodium aspartate,
3.6 KCl, 6 HEPES, 2.6 Tris, 0.7 CaCl2, 1 MgCl2, and 5 glucose. When
tetraethylammonium (TEA) or 4-aminopyridine (4-AP) was added to the
bathing solution, equimolar Na+
was replaced by these inhibitors. Because 4-AP is strongly basic, it
was neutralized by equimolar HCl. The pH values of all bath solutions
were finally adjusted to 7.4. Unless otherwise noted, KV currents were recorded when the
KIR currents were inhibited by 0.5 mM Ba2+. Higher concentrations of
Ba2+ were required for a complete
inhibition of KIR currents when the extracellular K+ concentration
was raised (see Fig. 3), probably because of the competition between
K+ and
Ba2+ (3).
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RESULTS |
Separation of KV current from
KIR current.
Both inward and outward currents were observed in all the cells
investigated. Ba2+ at a
concentration of 0.5 mM was able to inhibit the inward current almost
completely (Fig. 2). Inhibition of the
inward current is incomplete at lower concentrations (37). The reversal
potentials of the Ba2+-sensitive
current were
84.4 ± 0.8 mV and
85.7 ± 0.7 mV
(n = 26) for instantaneous and
steady-state currents, respectively. In addition, the steady-state
current-voltage relationship of the
Ba2+-sensitive current showed
inward rectification (c in Fig.
2B). The above characteristics of
the current sensitive to 0.5 mM
Ba2+ suggest that it belongs to
the category of KIR currents. The outward current sensitive to 0.5 mM
Ba2+ was regarded as being
mediated by KIR channels because
of its time course on depolarization from a holding potential of
90 mV. More specifically, the current reached its peak value
immediately after depolarization, indicating that the conductance
sensitive to 0.5 mM Ba2+ was
already activated at
90 mV. The above property is not likely to
be the case for KV currents. The
instantaneous peak current disappeared in the presence of 0.5 mM
Ba2+ (Fig.
2A). Similar outward currents
mediated by KIR channels have been
reported for other types of cells (18, 22). The slope conductance
between
100 and
60 mV, around the reversal potential of
the KIR current, was 93.0 ± 8.0 nS (n = 26) for the steady-state
KIR current (Fig.
2B).

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Fig. 2.
Separation of depolarization-activated outward K+
(KV) current from inwardly rectifying
K+
(KIR) current.
A: voltage protocol and current
recordings showing selective blockade of
KIR current by 0.5 mM
Ba2+. Series resistance (4.8 M )
was compensated by 90%. Leakage currents were subtracted. Arrowheads,
zero-current level; bar, 5 nA. B:
current-voltage relationship of peak currents resistant to 0.5 mM
Ba2+
(a) and instantaneous
(b) and steady-state
(c) currents blocked by 0.5 mM
Ba2+
(n = 26 for each data
set).
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The activation of the current resistant to 0.5 mM
Ba2+ was time and voltage
dependent (Fig. 2A). Both the
voltage dependency and time course of the current were apparently
different from those of the KIR
current. The slope conductance between
100 and
60 mV was
27.7 ± 2.5 nS (n = 26) for the
KV current (Fig.
2B). Further characterization of the
depolarization-activated outward current follows.
K+
selectivity, deactivation, and voltage-dependent activation.
Selectivity for K+ was examined
using the reversal potential
(Erev) of the
instantaneous tail current recorded after activation at 0 mV for 150 ms
(Fig. 3).
Erev was
85.3 ± 0.6 mV (n = 5) under control conditions (extracellular K+ concentration,
[K+]o = 3.6 mM) and shifted in a
positive direction when
[K+]o
was raised to 10 and 36 mM. The slope of the fitted line in Fig.
3C was 52.9 mV per 10-fold change in
[K+]o.
The above result indicates a high selectivity for
K+ in the
KV current.

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Fig. 3.
K+ selectivity and deactivation of
KV current.
A: voltage protocol and tail currents
under 2 different extracellular K+
concentration
([K+]o)
conditions. To block the KIR
current, 0.5, 1.5, and 5 mM Ba2+
were added to the bath solution when
[K+]o
was 3.6, 10, and 36 mM, respectively. Leakage currents were not
subtracted. Arrowheads, zero-current level; bars, 2 nA. Tail currents
were obtained after activation at 0 mV for 150 ms.
B: current-voltage relationship of
instantaneous tail currents shown in
A. C:
reversal potential
(Erev) of tail
currents plotted against
[K+]o
(number of observations in parentheses). Slope of straight line
obtained after applying linear regression was 52.9 mV/10-fold change in
[K+]o.
D: voltage protocol and tail currents
showing effects of Ba2+ on
deactivation. Bars, 2 nA. E: time
constant of deactivation
( deactivation) obtained from
single-exponential fittings to tail currents. Continuous lines are best
fits to linear regressions:
deactivation = 39.86 + 0.21Em for
control condition, and
deactivation = 10.62 + 0.06Em in
presence of 2 mM Ba2+.
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The time course of deactivation was apparently accelerated when both
[K+]o
and
[Ba2+]o
were elevated (Fig. 3A). To find the
reason for the above observation, the effect of
Ba2+ on deactivation under a
constant
[K+]o
condition was examined. As shown in Fig. 3,
D and
E, elevation of
[Ba2+]o
from 0.5 to 2 mM caused faster deactivation. Inhibition of full
activation by Ba2+ at this
concentration (see Fig. 8, B and
C) and/or a direct effect of
Ba2+ on the deactivation process
could be the mechanisms underlying the faster deactivation.
The activation of the outward K+
current was voltage dependent (Fig. 4). The
rising phase of the current was well fitted by two exponential
functions (Fig. 4B). The two time
constants (
ac.1 and
ac.2) at 0 mV determined from
the fitted curves were 7.9 and 58.8 ms, respectively (Fig.
4D). The time required for
half-maximal activation at 0 mV was 13.8 ± 3.4 ms
(n = 10). The threshold potential for
the activation of the KV
conductance was approximately
80 mV (Fig. 4,
C and
D). Maximal conductances
(gmax) were 154.1 ± 21.1 nS/cell or 6.0 ± 0.8 nS/pF (n = 9) for the peak current and 17.3 ± 4.1 nS/cell or 0.7 ± 0.2 nS/pF (n = 9) for the
steady-state current (Fig. 4C). The
normalized conductance calculated from the peak current was fitted by a
Boltzmann distribution, and the membrane potential
(Em) at the
half-maximal activation and the slope factor were estimated to be
42.6 mV and 17.5 mV/e-fold change in conductance, respectively (Fig.
4D).

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Fig. 4.
Activation of KV current.
A: voltage protocol and current
recordings. Leakage currents were subtracted. Bar, 5 nA. To secure
recovery from inactivation,
Em was held at
100 mV for 20 s between 2 test pulses.
B: fitting of double-exponential
functions to rising phase of currents presented in
A. Data for first 50 ms are shown.
First 0.6 ms was omitted from fitting because of uncompensated
capacitive currents. C: conductance
(g) plotted against membrane potential
(Em). Em values were
corrected for voltage errors derived from uncompensated series
resistance. As corrected
Em values were
different from cell to cell, all data points were plotted;
g was calculated using following
equation: g = I/(Em Erev),
where I is peak or steady-state
current at membrane potential of
Em and
Erev is reversal
potential for KV current
( 85.3 mV, Fig. 3C);
n = 9 for both peak and steady-state
currents. Fitted curves are g = 144.9/{1 + exp[( 47.6 Em)/14.2]}
for the peak conductance and g = 16.0/{1 + exp[( 76.15 Em)/6.0]}
for steady-sate conductance. Steady-state currents were obtained using
protocol shown in Fig. 5A.
D: normalized conductance
(g/gmax)
plotted against membrane potential
(Em);
gmax is maximal
conductance. Data presented in C were
used. Continuous curves are best fit to Boltzmann equation:
g/gmax = 1/{1 + exp[(E1/2 Em)/k]},
where E1/2 is
membrane potential at which one-half of channels are activated and
k is slope factor.
E1/2 and
k for peak current were 42.6 mV
and 17.5 mV/e-fold change in
conductance. E1/2
and k for steady-state current were
75.0 mV and 6.9 mV/e-fold
change in conductance. E: time
constants of activation
( activation) obtained from
double-exponential fittings as shown in
B (n = 10). Continuous lines for
ac.1 and
ac.2 are best fits to linear
and second-order regressions:
ac.1 = 7.866 0.044Em and
ac.2 = 58.798 0.030Em 0.003E2m.
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The activation curve of the steady-state component recorded using the
protocol shown in Fig.
5A was
also examined (steady-state data in Fig. 4,
C and
D).
Em at the
half-maximal activation and slope factor estimated by fitting a
Boltzmann distribution were
75.0 mV and 6.9 mV/e-fold change in conductance,
respectively. These values were apparently different from those of the
peak current.

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Fig. 5.
Inactivation of KV current.
A: voltage protocol and current
recordings. Leakage currents were subtracted. Arrowhead, zero-current
level; bar, 5 nA. To secure recovery from inactivation,
Em was held at
100 mV for 40 s between 2 test pulses.
B: fitting of double-exponential
functions to falling phase of currents presented in
A. Three test potentials were selected
for presentation. One-thirtieth of original data points are shown with
open symbols. C: current-voltage
relationship of peak currents and steady-state currents at end of 10-s
test pulses (n = 7).
D: time constants of inactivation
( inactivation) obtained from
double-exponential fittings as shown in
B (n = 7). Continuous lines for
inac.1 and
inac.2 are best fits to linear
regressions: inac.1 = 0.611 0.004Em,
and inac.2 = 4.255 0.016Em.
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Inactivation and recovery from inactivation.
The KV current was inactivated by
prolonged depolarization, but inactivation at steady state was
incomplete (Fig. 5A): steady-state currents at the end of 10-s voltage pulses normalized to the peak currents were 0.27 ± 0.01 (n = 7)
when Em was set
at 0 mV. The time course of inactivation was well fitted by two
exponential functions (Fig. 5B). The
two time constants (
inac.1 and
inac.2) at 0 mV determined
from the fitted lines were 0.6 and 4.3 s, respectively. Steady-state
inactivation was evaluated by a two-pulse protocol and by measuring
peak currents at +60 mV after 10-s prepulses (Fig.
6A).
Peak currents, normalized to the peak current after a prepulse of
100 mV, showed incomplete steady-state inactivation. The
voltage-independent fraction was estimated to be 0.21 from the fitted
curve (Fig. 6B).

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Fig. 6.
Steady-state inactivation of KV
current. A: voltage protocol and
current recordings. Leakage currents were subtracted. Arrowhead,
zero-current level; bar, 5 nA. To secure recovery from inactivation,
Em was held at
100 mV for 40 s before starting prepulses.
B: normalized peak currents
(I/Imax)
plotted against prepulse potential
(Eprepulse);
n = 10. Imax is peak
current when
Eprepulse is
100 mV. Continuous curve is best fit to following equation:
I/Imax = (1 S)/{1 + exp[(
Eprepulse E1/2)/k]},
where E1/2 is
membrane potential at which one-half of voltage-dependent fraction is
inactivated ( 53.4 mV), k is
slope factor (19.9 mV/e-fold change in
current), and S is voltage-independent
fraction (0.21).
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Recovery from inactivation was studied by varying the interval between
two test pulses (Fig.
7A). The
time required for complete recovery was dependent on the duration of
depolarization. Complete recovery from inactivation required ~20 s
after 0.5-s depolarization and 40 s after 10-s depolarization (Fig.
7B). The above result was taken into
account to determine the interval between voltage pulses in other
experiments presented in this study.

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Fig. 7.
Recovery of KV current from
inactivation. A: voltage protocol and
current recordings with a constant duration of test pulses
(tpulse) and
varied intervals between test pulses
(tinterval).
Leakage currents were subtracted. Arrowheads, zero-current level; bars,
2 nA. I0 and
I are peak currents elicited by first
and second voltage pulses, respectively.
B: normalized peak current
(I/I0)
plotted against
tinterval,
showing time course of recovery from inactivation
(n = 5) for each data point;
tpulse values
were set at 0.5 s ( ) and 10 s ( ). Continuous lines are best fits
to single-exponential functions:
I/I0 = 1 0.31exp( tinterval/5.67)
when tpulse = 0.5 s, and
I/I0 = 1 1.25exp( tinterval/9.18)
when tpulse = 10 s.
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Drug sensitivity and effect of
Ca2+ removal
from bath solution.
Quinine, verapamil, TEA, and Ba2+
inhibited both peak and steady-state currents dose dependently (Fig.
8).
IC50 values were estimated by
fitting the Hill equation (Fig. 8, B
and C).
IC50 values for verapamil were 4.8 × 10
5 M (peak) and
2.5 × 10
4 M (steady
state), for quinine were 3.8 × 10
5 M (peak) and 1.8 × 10
4 M (steady
state), and for TEA were 3.7 × 10
2 M (peak) and
>10
1 M (steady state).
The above results indicate that the peak current was more sensitive to
blockers than the steady-state current. From the dose-response
relationship, the effects of Ba2+
at concentrations <10
3 M
were considered to be relatively small. Although
IC50 values for
Ba2+ were not determined since
effects of Ba2+ at 0.5 mM cannot
be excluded, the IC50 for the peak
current is likely to lie between
10
3 and 5 × 10
3 M (Fig.
8B). The depolarization-activated
current was resistant to 4-AP even at a high concentration of 50 mM;
the difference between the current magnitude under control conditions
and that in the presence of 50 mM 4-AP was not statistically
significant (P > 0.05 with
Student's t-test) for both peak and
steady-state currents (n = 5).

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Fig. 8.
Effect of inhibitors and removal of extracellular
Ca2+ on
KV current.
A: voltage protocol and current
recordings. Leakage currents were subtracted. Arrowheads, zero-current
level; bars, 5 nA. To secure recovery from inactivation,
Em was held at
100 mV for 40 s before starting test pulses. All currents were
recorded under inhibition of KIR
currents by 0.5 mM Ba2+. Currents
in presence of inhibitors were obtained when currents reached a steady
level after 3-4 test pulses.
Ca2+-free bath solution contained
1 mM EGTA. B and
C: dose-response relationships of
normalized peak currents (B) and
steady-state currents at end of 10-s voltage pulses
(C)
(n = 5 for each data point). Curves
are best fits to Hill equation:
I/I0 = 1 [inhibitor]n/(ICn50 + [inhibitor]n),
where I0 is
current under control condition and n
is Hill coefficient. The n values for
peak currents were 1.2 (verapamil), 1.5 (quinine), 2.1 [tetraethylammonium (TEA)], and 3.5 (Ba2+). The
n values for steady-state currents
were 0.99 (verapamil) and 1.3 (quinine).
D: effect of
Ca2+ removal on activation of peak
currents. Activation curves were obtained in same way as in Fig.
4D. Data were normalized to maximal
conductance (gmax) under
control condition (n = 4). Curves are
best fits to following equation:
g/gmax
of control = a/{1 + exp[(E1/2 Em)/k]}.
E1/2,
a, and
k for control condition are
43.0 mV, 1, and 17.1 mV/e-fold
change in conductance.
E1/2,
a, and
k for
Ca2+-free condition are
50.5 mV, 1.45, and 17.1 mV/e-fold change in conductance. 4-AP,
4-aminopyridine.
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To exclude the possibility that
Ca2+ flowed into the cell on
depolarization and activated the relevant
K+ channel, extracellular
Ca2+ was removed and 1 mM EGTA was
added to the bath solution. As shown in Fig.
8A, an increase rather than decrease
in the peak current by 49.8 ± 7.9%
(n = 9) was observed. The above result and the relatively strong chelation of intracellular
Ca2+ by 5 mM EGTA make it unlikely
that the depolarization-activated K+ current was activated by
intracellular Ca2+. To elucidate
the reason for the increase in the peak current under the
Ca2+-free condition, effects of
Ca2+ removal on activation were
examined (Fig. 8D).
Ca2+ removal caused an increase in
the maximal conductance by 45% and a change in
Em at
half-maximal activation
(E1/2) by
7.5 mV. Liberation from a charge-screening effect of
Ca2+ could be a cause of the
change in E1/2,
but the apparent increase in the maximal conductance could not be
explained by the above mechanism. Extracellular
Ca2+ might block the
KV channel as reported for other
voltage-dependent K+ channels (14,
15). The reversal potential of tail currents under the
Ca2+-free condition (
85.1 ± 0.8 mV, n = 5) obtained using
the protocol in Fig. 3A was not
significantly different from that under control conditions. Thus a
decrease in the K+ selectivity of
KV currents in a
Ca2+-free medium reported for
squid neurons (2) and/or a substantial contribution from a
voltage-activated inward Ca2+
current under control conditions could be excluded.
Em under zero-current
clamp conditions.
To assess the possible contribution of
KV conductance to
Em, the effects
of 0.5 mM Ba2+ and 1 mM quinine
were examined in the zero-current clamp mode (Fig.
9).
Ba2+ at 0.5 mM was used to block
the KIR conductance almost
completely (Fig. 2), and 1 mM quinine was used to block both peak and
steady-state components of KV
conductance (Fig. 8).
Em under control
conditions was
87.1 ± 1.3 mV
(n = 8), which was close to the
equilibrium potential for K+ (EK;
93 mV). Depolarization to
76.4 ± 3.6 mV
(n = 8) occurred when the
KIR conductance was inhibited by
0.5 mM Ba2+. Inhibition of both
KIR and
KV conductances by 0.5 mM
Ba2+ and 1 mM quinine caused
further depolarization to
12.5 ± 4.0 mV
(n = 8). A selective and complete
inhibition of the KV conductance without effects on the KIR
conductance could not be achieved, as we have not found a selective
blocker of the KV conductance in
the intermediate cell. Although quinine and verapamil inhibited both
peak and steady-state components of the
KV current almost completely (Fig.
8), these blockers also inhibited the
KIR current (37).

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Fig. 9.
Em recorded
in zero-current clamp mode. A:
representative
Em trace
showing effects of 0.5 mM
Ba2+ and 1 mM quinine.
B: summary of effects of
Ba2+ and quinine on
Em
(n = 8). Data were obtained when
Em reached
a steady level 20-40 s after a solution change.
* Statistically significant difference from control based on
paired Student's t-test
(P < 0.05).
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DISCUSSION |
Method for separation of the KV current
from the KIR current.
In all patch-clamped intermediate cells, the
KIR current was observed under the
experimental conditions described in this study. Thus elimination of
the KIR current was essential for
the characterization of the KV
current. It has been reported that KIR currents are more sensitive to
Ba2+ than
KV currents (27). The
IC50 of
Ba2+ for the
KIR current in the intermediate
cell has been shown to be
~10
5 M (37), whereas that
for the KV current is likely to
lie between 10
3 and
10
2 M (Fig. 8). Incomplete
suppression of the KIR current is
not likely, since currents at
Em values lower
than the EK
(
93 mV) were essentially null
(a in Fig.
2B).
Substantial inhibition of the KV
current by 0.5 mM Ba2+ is not
likely, since apparent effects of
Ba2+ on this current only appeared
at higher concentrations (>1 mM, Fig. 8). Among all the methods
tested, the application of 0.5 mM
Ba2+ to the bath solution was the
only effective method for the selective and complete inhibition of the
KIR current. For example,
extracellular Cs+, which is known
to inhibit KIR currents, blocked
the inward current but failed to block the outward current, which we
regarded as being mediated by the
KIR channel (see Fig. 4 in Ref.
37). Removal of extracellular K+
reduced not only the KIR current
but also the KV current. Removal of ATP from the pipette solution was ineffective for the suppression of
the KIR current.
Characteristics of the KV current in the
intermediate cell.
Several characteristics of the KV
current in the intermediate cell, such as voltage-dependent activation,
speed of activation and inactivation, and independence from the
intracellular Ca2+, resemble those
of delayed rectifier K+ currents,
whereas the time courses of activation fitted by double-exponential functions were different from those of delayed rectifier
K+ currents that are known to be
fitted by sigmoidal functions (28). One of the distinctive
characteristics of K+ conductance
is its activation at relatively deep negative
Em values.
Outward K+ currents activated in a
similar Em range
have been reported (16, 17, 29). Although the majority of cloned
voltage-activated K+ channels are
activated at more depolarized
Em values, a
modification of channel properties may occur in native cells. In
relation to the above discussion, it has been reported that cell
culture causes a positive shift of activation potential (45).
Incomplete inactivation even at the end of a 10-s depolarization is
another characteristic of the KV
current in the intermediate cell. Similar incomplete inactivation has
been reported for KV currents in
native cells (17, 23, 29) and those mediated by cloned
KV channels (10, 11, 31, 44).
When the steady-state current was analyzed separately, the activation
curve was quite different from that of the peak current (Fig.
4D). The above result and the
difference in the drug sensitivity between the peak current and the
steady-state current (Fig. 8) raise the possibility that the
KV current in the intermediate cell is derived from two distinctive channels, although it cannot be
excluded that peak and steady-state currents might be derived from
different states of one channel.
The ineffectiveness of 4-AP at 50 mM and the relatively high
IC50 for TEA (37 mM for the peak
current) also characterize the KV
current in the intermediate cell, since the majority of cloned KV channels are sensitive to 4-AP
and/or TEA at lower concentrations (24). The above characteristic does
not necessarily imply that a large structural difference exists between
known KV channels and the relevant
K+ channel, since even small
differences in the molecular structure may have significant effects on
certain properties of the channel. For example, it has been reported
that KV channels belonging to the
same subfamily but isolated from different animal species differ in
certain properties such as drug sensitivity (1).
Possible roles of KV conductance in the
intermediate cell.
It is known that KV currents
regulate the duration of action potential in excitable cells (13). In
nonexcitable cells, such currents have also been reported (e.g., Refs.
8, 35, 41). With regard to the physiological function of
K+ channels in the intermediate
cell, they have been proposed to contribute to the generation of
positive EP (32, 42). The KIR
channel reported in the previous study was suggested to contribute to
EP (37). Because KV conductance is
activated by depolarization, its contribution to
Em depends on the
relative magnitude of other conductances in the intermediate cell,
which remain unknown except for
KIR conductance.
To discuss the Em
of the intermediate cell in vivo,
K+ concentration in the
intercellular space of the stria vascularis, where the intermediate
cell is found, should be considered. Although a general agreement on
the above-mentioned K+
concentration has not been arrived at because of technical
difficulties, a low concentration comparable to that found in usual
extracellular fluids has been suggested (30). Several lines of research
on the
Na+-K+-ATPase
(5, 19, 32) and the
Na+-K+-Cl
cotransporter (25, 38, 42) in the basolateral membrane of the marginal
cell support the putative low concentration in the intercellular space,
because these transporters are thought to take up
K+ and keep
K+ concentration in the
intercellular space low (Fig. 1). The
Na+-K+-ATPase
in the intermediate cell (5, 19) may also contribute to taking up
K+ from the intercellular space.
Assuming usual intracellular and extracellular
K+ concentrations,
EK would be
expected to lie in the range between
80 and
95 mV. The
above discussion and the activation curve of
KV conductance (Fig.
4D) suggest that a slight
depolarization can activate KV
conductance even if the
Em of the
intermediate cell in vivo is close to
EK. The activated
KV channel stabilizes the
Em near
EK. Incomplete
inactivation of KV conductance on
prolonged depolarization may be favorable for stabilizing
Em in case of prolonged depolarization.
The above speculation was examined by studying
Em in the
zero-current clamp mode (Fig. 9). When
KIR conductance was blocked completely by 0.5 mM Ba2+,
depolarization by 10.7 mV occurred. Thus
KIR conductance was indispensable
for keeping Em
near EK. However,
Em was still
maintained at a relatively deep negative value of
76.4 mV when
KIR conductance was blocked almost
completely. It is very likely that
Em in the presence of 0.5 mM Ba2+ was
maintained by KV conductance.
Although the slope conductance between
100 and
60 mV of
the KV current was ~30% of that
of the KIR current (Fig.
2B), the size of the
KV conductance may be sufficient
to maintain the relatively deep negative
Em.
The second possible function could be to provide a pathway for
K+ secretion.
K+ secretion into the
intercellular space in the stria vascularis is important as part of the
K+-recycling pathway from the
perilymph to the endolymph (20, 32, 33).
K+ secreted into the intercellular
space is thought to be taken up by the marginal cell via
Na+-K+-ATPase
and the
Na+-K+-Cl
cotransporter, as mentioned above, and finally secreted into the
endolymph. As the KIR channel in
the intermediate cell is likely to mediate outward currents at
Em values near
EK (see
b and
c in Fig.
2B), the
KIR channel is one possible
pathway for K+ secretion. In
addition, when the
Em of the
intermediate cell in vivo is above the activation threshold of the
KV channel, it would provide an
additional route for K+ secretion
into the intercellular space. The incomplete inactivation on prolonged
depolarization may favor the open state of this additional route. The
above-mentioned supportive role of the
KV channel may be important when
the activity of the KIR channel declines.
Finally, the KV channel may be
involved in cell proliferation reported for the intermediate cell of
adult guinea pigs (6) because KV
channels have been proposed to be involved in mitosis of several types
of cells (9, 26, 46).
 |
ACKNOWLEDGEMENTS |
We thank Professor Akihiko Irimajiri for his encouragement and
Takako Ichinowatari for her secretarial work.
 |
FOOTNOTES |
This work was supported by Grants 9671749 and 10770889 from the
Ministry of Education, Science, Sports and Culture, Japan.
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: S. Takeuchi,
Dept. of Physiology, Kochi Medical School, Nankoku 783-8505, Japan
(E-mail: takeuchi{at}kochi-ms.ac.jp).
Received 2 October 1998; accepted in final form 7 April 1999.
 |
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