A cationic nonselective stretch-activated channel in the
Reissner's membrane of the guinea pig cochlea
Te-Huei
Yeh1,2,
Philippe
Herman1,
Ming-Cheng
Tsai3,
Patrice Tran Ba
Huy1, and
Thierry
Van Den
Abbeele1
1 Laboratoire d'Otologie
Experimentale, Faculté de Médecine
Lariboisiére- Saint Louis, 75010 Paris, France; and
2 Department of
Otolaryngology, National Taiwan University Hospital, and
3 Department of
Pharmacology, National Taiwan University College of Medicine,
Taipei, Taiwan, Republic of China
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ABSTRACT |
The Reissner's membrane (RM) separates in the mammalian cochlea
the K+-rich endolymph from the
Na+-rich perilymph. The
patch-clamp technique was used to investigate the transport mechanisms
in epithelial cells of RM freshly dissected from the guinea pig
cochlea. This study shows a stretch-activated nonselective cationic
channel (SA channel) with a linear current-voltage relationship (23 pS)
highly selective for cations over anions [K+
Na+ (1) > Ba2+ (0.65) > Ca2+ (0.32)
Cl
(0.14)] and
activated by the intrapipette gradient pressure. The open
probability-pressure relationship is best fitted by a Boltzmann
distribution (half-maximal pressure = 37.8 mmHg, slope constant = 8.2 mmHg). SA channels exhibit a strong voltage dependency and are
insensitive to internal Ca2+, ATP,
and fenamates but are blocked by 1 µM
GdCl3 in the pipette. They are
reversibly activated by in situ superfusion of the cell with hyposmotic
solutions. Kinetic studies show that depolarization and mechanical or
osmotic stretch modify the closed and open time constants probably by a
different mechanism. These channels could participate in
pressure-induced modifications of ionic permeability of the RM.
patch-clamp technique; ion channels; endolymphatic
hydrops
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INTRODUCTION |
REISSNER'S MEMBRANE (RM) is a thin structure made of
two cell layers that separates the scala vestibuli from the scala media in the mammalian cochlea. These two fluid-filled compartments differ
dramatically in their ionic composition. The epithelial cell layer of
RM is made of confluent epithelial cells and faces the scala media,
which contains endolymph, a fluid rich in
K+ (the major ion involved in
sensory mechanotransduction). In contrast, the mesenchymal cell layer
is made of nonconfluent conjunctival cells and faces the scala
vestibuli, which contains perilymph, a plasmalike fluid rich in
Na+.
Although the exact nature of its role remains uncertain, the RM is
thought to be a barrier involved in maintaining the asymmetrical electrolytic compositions of the two fluids. In Ménière's
disease, a major cause of hearing loss in humans, the RM appears to
become progressively distended by endolymphatic hydrops, an increase in
the volume of the endolymph compartment, and the main symptoms are
attributed to a leak of K+ from
the endolymph to the perilymph. Endolymphatic hydrops is probably the
result of an imbalance between the production and absorption of the
endolymph, leading to a gradual increase in volume. The membranous
labyrinth is surrounded by an inextensible bony envelope in mammals,
which would increase the endolymphatic pressure. Changes in the
intralabyrinthic pressure in guinea pigs by endolymph infusion (24) or
blockade of the vestibular aqueduct (1, 13) gradually distends the RM.
This distension is thought to alter the ionic permeability of the RM,
as suggested by changes in the electrochemical composition of the
endolymph in humans (26) and in an experimental model (22). However,
very little is known of the ion transport mechanisms involved in the
RM, partly because its delicacy and inaccessibility render studies
difficult.
This study used a novel preparation of freshly dissected RM isolated
from the guinea pig cochlea. The luminal side of the epithelial cells
facing the endolymph was investigated by the patch-clamp technique in
the cell-attached and inside-out configurations. The most frequent
channel was a stretch-activated channel (SA channel) observed in
cell-attached and excised patches. A
Ca2+-activated channel (CAN
channel) was also observed in some excised patches. The two channels
had similar conductive properties but differed in their sensitivity to
blockers, voltage, and activators. These channels could account for the
homeostasis of the endolymph under physiological conditions and the
possible ionic changes of this fluid during endolymphatic hydrops.
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METHODS |
Tissue preparation.
Young male pigmented guinea pigs (250-300 g, Elevage d'Ardenay,
France) were given a lethal dose of anesthetic (100 mg/kg pentobarbital) and decapitated. The bullae were immediately removed and
immersed in ice-cold culture medium (L15-Leibowitz, Eurobio, Les Ulis,
France). The RM were dissected out as described by Yeh et al. (29).
Briefly, fragments of RM were identified, under a dissecting
microscope, dissected free with a sharp needle, and placed in culture
medium (Fig.
1A).
The two-cell-layer section was examined at high magnification, and the
epithelial side was identified by its higher density of nuclei compared
with the perilymphatic side (28). Fragments were then placed epithelial
side up, on a petri culture dish with the tip of a needle (Falcon,
Polylabo, Paris, France).

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Fig. 1.
Patch-clamp recording of the Reissner's membrane.
A: photograph showing piece of
Reissner's membrane that was dissected out and placed on a petri dish
bottom with a patch-clamp pipette. Epithelial cells are polyhedral with
a dark cytoplasm and a round nucleus, whereas nuclei of mesenchymal
cells are irregular and dispersed. B:
diagram describing preparation and recording procedures.
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Patch-clamp recordings and analysis.
Single-channel currents were recorded with a RK400 patch-clamp
amplifier (Biologic, Claix, France) from patches of apical membranes of
epithelial cells of RM using the cell-attached and inside-out
configurations of the patch-clamp technique (4). Patch pipettes (tip
resistance 8-15 M
) were made from microhematocrit capillary
tubes (CHR Badram, Bizkerod, Denmark) and coated with Sylgard (Dow
Corning, Seneffe, Belgium). Pressure in the lumen of the pipette was
generated and monitored by a pneumatic tester (Interacoustics AZ7R,
Assens, Denmark) and ranged from
60 to +40 mmHg. Signals were
filtered at 500 Hz with an eight-pole Bessel filter (Frequency Devices,
Haverhill, MA) and digitized at 1 kHz using a Labmaster analog-digital
interface and Acquire software (Biologic). Liquid junction potentials
were determined separately, and potentials were corrected accordingly.
Single-channel current recordings were analyzed with custom-written
software. Inward currents flowing from the pipette into the cell are
shown as downward deflections. The mean current
(I) passing through
N (number of channels in the patch,
estimated from current amplitude histograms) channels was used to
calculate NPo or
Po using the
equation I = NPoi,
where Po is the
open probability and i is the unit
current. I was calculated as the
integral of the currents, taking the closed level as a baseline.
Because the channels usually had a low resting
Po in the resting
cell-attached condition, the closed level current was determined at the
beginning of each recording.
The Goldman-Hogkin-Katz modified constant field equation was used to
estimate the current reversal potential and then the relative
permeabilities of the cationic channels,
PK/PNa,
PK/PCl, PBa/PK,
and
PCa/PK.
The Levenberg-Marcquardt algorithm (Sigmaplot 2.0, Jandel) was used to
fit the
Po-membrane
voltage (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. The
Po-pressure
relationship was fitted by the same type of relationship
where
Pmax is the
maximal Po and
P50 is the half-maximal pressure.
All the kinetic analyses were performed on patches containing
apparently a single activated channel. Idealized recordings were
obtained by a half-amplitude threshold method, after analog filtering
to avoid spontaneous peaks on the baseline exceeding the detection
threshold. Intervals were then measured, binned, and fitted to the sum
of exponentials using the maximum likelihood method (Biopatch analysis
software, Biologic). The dwell-time distribution was fitted to the
following function:
N(t) = A ·
+ B ·
, where N is the number of events,
A and
B are the coefficients, and
1 and
2 are the slow and fast time
constants, respectively.
Experimental values are given as means ± SE, and
n is the number of successful
recordings.
Solutions.
In the cell-attached configuration, the pipette solution mimicked the
endolymph and contained (in mmol/l) 145 KCl, 1.2 MgCl2, 1 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) adjusted to pH 7.4 with KOH, or NaOH for some experiments in which 145 KCl was replaced by 145 sodium gluconate, 90 BaCl2, or 90 CaCl2. The standard bath solution
was a perilymph-like solution containing (in mmol/l) 140 NaCl, 4.8 KCl,
1.2 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES
adjusted to pH 7.4 with NaOH. In some experiments in the cell-attached
configuration, the cells were superfused with hypotonic bath solution
containing 100 mmol/l NaCl instead of 140 mmol/l NaCl. In excised
inside-out patch experiments, low-Ca2+ standard bath solutions
were buffered at 10
9 mol/l
CaCl2 by 2 mmol/l ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) without addition of 1 mmol/l
CaCl2. Flufenamic acid and ATP
were obtained from Sigma France, and gadolinium chloride was from
Prolabo (Paris, France).
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RESULTS |
Channel activities in cell-attached configuration and effects of
pipette pressure.
Most of the successful patches (n = 117 of 176, 66%) contained spontaneously active channels, with rare,
brief openings at positive pipette potentials and longer openings at
negative potentials (see Fig.
2A for a
typical recording). The
Po of the
channels was increased when suction was applied to the cell-attached
patches. The delay of activation was shorter than 1 s, and the first
detectable activity occurred usually near 20 mmHg. The channels were
deactivated immediately after release of the pressure. The density of
these mechanosensitive channels was high (6.5 ± 0.8 per patch,
n = 35). In contrast, none of the 18 patches made on mesenchymal cells had any stretch-sensitive activity. A
positive pressure gradient activated the channels in much the same way
as a negative one, but gigaseals were more difficult to maintain.
Figure 3A
shows typical responses of these mechanosensitive channels to stepwise increases in pressure, and Fig. 3B
shows the relationship between Po and pressure.
The related data points were best fitted by a sigmoidal curve adjusted
to a Boltzmann distribution with a detectable increase of activity near
15 mmHg. The half-maximal pressure was 37.8 mmHg, and
d was equal to 8.2 mmHg. In these
measurements, the pipette pressure was maintained for ~5-10 s.
The pressure gradient was held constant for several minutes in some
longer recordings, without inactivation of the channels (data not
shown). The maximal
Po never exceeded
0.8, even when a high pressure gradient or a strong depolarization was
applied to the patch, probably because the channels had a flickering
activity with many closed time periods. The resting
Po and the
sensitivity to stretch remained unchanged when the patches were excised
and placed in a low-Ca2+ standard
bath (2 mmol/l EGTA without CaCl2,
Fig. 3B).

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Fig. 2.
Voltage dependence of stretch-activated nonselective cationic channels
(SA channels) with and without pressure.
A: representative single-channel
recordings obtained from a cell-attached patch at various voltages. No
pressure was applied to the pipette.
Vm, membrane
voltage; Po, open
probability; C, closed state. B:
Po values at
various voltages of 7 different patches containing SA channels. No
pressure was applied to the pipette. Line is the least-squares fit to
the Boltzmann relationship (see
METHODS for details). Half-maximal
voltage was +7 mV. A depolarization of 24 mV was needed to increase
Po by an
e factor.
C: 3-dimensional mesh diagram of the
relationship between negative intrapipette pressure,
Vm, and
Po. Sixteen
points indicate the mean
Po of 3 cell-attached patches held at 4 voltages ( 60, 30, +30,
and + 60 mV) and 4 negative pressures (0, 20, 35, and 45 mmHg). This
diagram shows that depolarization increased the sensitivity to
pressure, since half-maximal pressure was reduced, but did not alter
the maximal Po.
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Fig. 3.
Dependence of single-channel activity on pipette suction. Pipette
solution contained 145 mmol/l KCl, bath solution contained 140 mmol/l
NaCl solution, and
Vm was clamped at
60 mV. A: single-channel
currents from a cell-attached patch at different pipette suction
pressures (solid bar). C and bars
(left) indicate closed state
current, and numbers (right)
indicate the negative pressure (mmHg).
B: relationship between
Po and applied suction (mmHg). ,
Po values (means ± SE) of 8 cell-attached patches. ,
Po values (means ± SE) of same excised inside-out patches in a
low-Ca2+ standard solution (2 mmol/l EGTA). Clamp potential
(Vc) was
60 mV. Data points were best fitted by the modified Boltzmann
equation using the Marcquardt method of least squares (see
METHODS for details). Half-maximal
pressures in cell-attached (37.8 mmHg) and inside-out (42.2 mmHg)
configurations were similar. A detectable increase in SA channel
activity was observed at 10-20 mmHg.
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Unitary conductance and ion selectivity.
The SA channels in cell-attached patches bathed in standard bath
solution, with KCl in the pipette, displayed a unit conductance of 23.7 ± 0.4 pS (n = 24, Fig.
4A). The
reversal potential
(Er) was not
significantly different from 0 mV, suggesting that these cells had a
very low resting potential. The conductive properties of these channels
were then studied in inside-out configuration, with the bath containing
various solutions with low-Ca2+
concentration to eliminate the activity of
Ca2+-dependent nonselective
channels (see Fig. 6A) and a minimal pressure of 30 mmHg to maintain a significant
Po. The
conductance and the Er remained
identical after excision in inside-out configuration in a bath
containing a standard solution (21.8 ± 0.4 pS,
Er = +4.9 mV,
n = 18, Fig.
4C) or 145 mmol/l KCl (23.9 ± 0.4 pS, Er =
1 mV, n = 32, Fig.
4A). Dilution of the KCl bath from
145 to 42 mmol/l shifted the
Er to +22.3 ± 0.2 mV (n = 6, Fig.
4B), very near to the Nernst
equilibrium potential for cations. These results indicate that the
channels were less permeable to anions than cations
(PK/PCl = 7) and did not discriminate between monovalent cations
(PK/PNa = 1.2). The unit conductance and
Er values (21.2 ± 0.4 pS and
3.5 mV, respectively,
n = 6, Fig.
4D) and the
Po-pressure relationships (data not shown) of cell-attached and inside-out patches
were not affected when the pipette contained 145 mmol/l of potassium
gluconate. This indicates that the SA channels detected in the
cell-attached configuration and inside-out patches are identical.

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Fig. 4.
Ion selectivity. Current-voltage
(i-V)
relationships of channels in cell-attached and excised inside-out
patches in a low-Ca2+ standard
solution (2 mmol/l EGTA).
Vc was identical
to Vm for
inside-out patches but represents
Vm Em for the
cell-attached patches, in which the spontaneous membrane potential
(Em) is
unknown. A: pipette solution contained
145 mmol/l KCl. , Current values (means ± SE) of 24 cell-attached patches. , Current values (means ± SE) of 32 excised inside-out patches, with bath containing 145 mmol/l KCl. Unit
conductances were 23.7 (cell-attached) and 23.9 (inside-out) pS, and
reversal potential was near 0 mV. B:
cation vs. anion selectivity in inside-out configuration. Pipette
solution contained 145 mmol/l KCl, and bath solution was diluted to 42 mmol/l KCl. Reversal potential was shifted to +22.3 ± 0.2 mV
(n = 6), close to the Nernst
equilibrium potential for cations. Ratio for relative permeabilities of
K+ and
Cl
(PK/PCl)
was 7. C:
Na+ substitution in inside-out
configuration. Bath contained standard solution with 140 mmol/l NaCl,
and pipette contained 145 mmol/l KCl. The
i-V
relationship remained linear, with a reversal potential at +4.9 mV,
indicating poor discrimination between
K+ and
Na+
(PK/PNa = 1.2). D: gluconate substitution in
inside-out configuration. Pipette solution contained 145 mmol/l of
potassium gluconate instead of KCl. Reversal potential was near 0 mV,
indicating that gluconate did not permeate the channel. Error bars are
indicated when SE exceeded the size of the symbols. Lines in
A, C,
and D are linear regressions of the
points weighted at each voltage by the SE. Line in
B is the least-squares fit to the
Goldman relationship.
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Permeability to divalent cations.
The permeability of the SA channel to divalent cations was determined
by replacing the content of the pipette with 90 mmol/l BaCl2 or 90 mmol/l
CaCl2 (Fig.
5A).
BaCl2 reduced the unit conductance to 13.4 ± 0.5 pS (n = 7), and
CaCl2 reduced it to 14 ± 0.6 pS (n = 8). The current-voltage
relationship showed a slight rectification that could be fitted by a
Goldman equation. The
Er value was
slightly shifted to
5.2 mV with
BaCl2 and to
8 mV with
CaCl2. The
PBa/PNa ratio calculated from the modified constant field equation was 0.65, and the
PCa/PNa
ratio was 0.32. These values indicate a significant permeability for
divalent cations. Figure 5B shows sigmoidal
Po-pressure
relationships like those obtained with monovalent cations in the
pipette, but the half-maximal pressures were shifted to 28.4 and 28.8 mmHg, respectively.

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Fig. 5.
Permeability to divalent cations. A:
i-V
relationships of the SA channels in inside-out patches. Pipette
solution contained 90 mmol/l BaCl2
( , n = 7) or 90 mmol/l
CaCl2 ( ,
n = 5), and bath contained a control
standard solution. Symbols represent current values (means ± SE),
and lines are the first-order regression lines, yielding unit
conductances of 13.4 pS (Ba2+)
and 14 pS (Ca2+) and a reversal
potential of 5.2 mV
(Ba2+) and 8 mV
(Ca2+). This indicates a lower,
but still significant, permeability to divalent cations than to
monovalent cations
(PBa/PNa = 0.65 and
PCa/PNa = 0.32). B: relationship between
Po and applied
suction (mmHg). ,
Po values (means ± SE) of 7 excised inside-out patches with 90 mmol/l
BaCl2 inside the pipette. ,
Po values (means ± SE) of 5 excised inside-out patches with 90 mmol/l
CaCl2 inside the pipette.
Vc was 60
mV. Data points were best fitted by the modified Boltzmann equation
using the Marcquardt method of least squares (see
METHODS for details). Half-maximal
pressures in cell-attached (28.4 mmHg) and inside-out (28.8 mmHg)
configurations were similar. A detectable increase in SA channel
activity was observed at 10-20 mmHg.
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Voltage dependence.
Figure 2A shows typical recording
traces at various voltages, showing that the activity of the SA
channels strongly depends on the voltage, even without any negative
pressure in the pipette. The relationship between
Po and voltage
was sigmoidal and followed a Boltzmann distribution (Fig.
2B). Even when strong
depolarizations were applied to the patches, the
Po did not exceed
0.8, probably due to the flickering activity of the channel (see the
recording at +60 mV, Fig. 2A). In
the absence of negative pressure, the V50 was +7 mV and
a depolarization of 24 mV increased the
Po by an
e-fold factor. When the pressure in
the pipette was negative, the half-maximal potential was shifted toward
more negative values and the slope was reduced (Fig.
2C).
Effects of internal
Ca2+ and
blockers.
The spontaneous activity of some patches (22 of 63 patches, 35%) was
increased when patches were excised in inside-out configuration in a
standard saline solution containing 1 mmol/l
CaCl2. These channels had a
conductance near 25 pS, similar to SA channels (see Fig.
6A), and
became inactive when the internal side of the membrane was exposed to
low-Ca2+ standard solution (2 mmol/l EGTA, n = 5) or 1 mmol/l ATP
(n = 3). The addition of a negative
pressure gradient in the pipette revealed the presence of SA channels
with a flickering behavior in all these conditions. The slope and the
half-maximal pressure of the
Po-pressure
relationship were independent of the internal Ca2+ concentration (Fig.
6A). Figure
6B shows the absence of effect of
flufenamic acid, a blocker of
Ca2+-activated nonselective cation
channels, on the SA channel in the absence of intracellular
Ca2+. These results indicate that
SA channels are not affected by blockers or activators of
Ca2+-activated nonselective
cationic channels and thus are probably distinct from the family of CAN
channels described in many epithelial tissues, including outer hair
cells (27) and native (23) and cultured marginal cells (30).

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Fig. 6.
Insensitivity of the SA channel to internal
Ca2+ and flufenamic acid. Pipette
solution contained 145 mmol/l KCl, bath contained 140 mmol/l NaCl
solution, and Vm
was clamped at 60 mV A:
relationship between
Po and applied
suction (mmHg) in inside-out configuration. ,
Po (means ± SE) of 5 inside-out patches when the bath contained 1 mmol/l
CaCl2. ,
Po (means ± SE) of same patches when the bath contained 2 mmol/l EGTA. Data points
were best fitted by the modified Boltzmann equation using the
Marcquardt method of least squares (see
METHODS for details). Both curves are
parallel with a threshold to pressure near 20 mmHg and half-maximal
pressures of 32.6 and 40.6 mmHg, respectively. Recordings
(bottom) were obtained from the same
patch with 1 mmol/l CaCl2
(top recording) and with 2 mmol/l
EGTA (bottom recording). C, closed
level. In presence of internal
Ca2+, one level of
Ca2+-activated channel showed
prolonged opening, whereas the SA channels showed a flickering
behavior. B: relationship between
Po and applied
suction (mmHg) in inside-out configuration. ,
Po (means ± SE) of 4 inside-out patches when the bath contained 1 mmol/l flufenamic
acid without CaCl2. ,
Po (means ± SE) of same patches without flufenamic acid. Data points were best
fitted by the modified Boltzmann equation using the Marcquardt method
of least squares (see METHODS for
details). No significant difference exists between the curves. A
detectable increase in SA channel activity was observed near 20 mmHg,
and half-maximal pressures were 46.6 and 54.4 mmHg, respectively.
Recordings (bottom) were obtained
from same patch with 1 mmol/l flufenamic acid (top
recording) and without flufenamic acid
(bottom recording).
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In contrast, Gd3+, a blocker of SA
channels in a variety of tissues, was a potent blocker when applied to
the extracellular side of the membrane. The maximal pressure of the
Po-pressure relationship in Fig. 7A
was greatly reduced by 1 µmol/l
GdCl3 in the pipette, and the
half-maximum pressure was shifted from 37.7 to 45.7 mmHg.
Gd3+ reduced the
Po, but the
unitary conductance of the channel remained unchanged. Higher
concentrations of GdCl3 (1 mmol/l)
were required to completely block the SA channels when it was applied
to the internal side of the membrane. This blockade was slowly reversed after washout of the Gd3+ (see
Fig. 7B).

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Fig. 7.
Blockade of the SA channels by internal and external gadolinium.
A: relationship between
Po and applied
suction (mmHg) in inside-out configuration. ,
Po (means ± SE) of 7 inside-out patches. ,
Po (means ± SE) of the 5 patches when pipette was filled with 1 µmol/l of
GdCl3. Bath contained a
low-Ca2+ standard solution (2 mmol/l EGTA). Data points were best fitted by the modified Boltzmann
equation using the Marcquardt method of least squares (see
METHODS for details). Half-maximal
pressures were 37.7 and 45.7 mmHg, and slope constants were 8.3 mmHg
and 13.7 mmHg, respectively B: typical
traces of an excised inside-out patch with and without internal
gadolinium (1 mmol/l) with 2 mmol/l EGTA. Pipette solution contained
145 mmol/l KCl, bath contained 140 mmol/l NaCl solution, and
Vm was clamped
at 60 mV.
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In situ activation of the SA channel by hyposmotic-induced swelling.
The sensitivity to mechanical stretch of the channels described here
and the well-known influence of osmotic and pressure changes in the
inner ear on the homeostasis of the endolymph suggested that these
channels may also be activated by osmotic pressure. This was
investigated using the cell-attached configuration to study the in situ
effects of hyposmotic-induced swelling. Superfusion of the cells with
hyposmotic standard (~200 mosmol/l) solution increased the activity
of the channels in three of four patches with a mean delay of 28 ± 5 s (n = 3). In these three cases, the NPo before and
during the superfusion of the hypotonic solution increased (0.17 ± 0.07 to 0.97 ± 0.24). This effect was reversible after washout with
the standard solution
(NPo = 0.13 ± 0.07) with a similar delay. The number of SA channels was assessed
before and after the experiment by applying a negative pressure to the pipette (average of 7.3 channels activated by maximal pressure in the 3 responding patches). Figure 8 shows a
typical single-channel current recording from a cell-attached patch
containing SA channels sensitive to mechanical stretch and osmotic
pressure. The flickering activity of SA channels activated by
intrapipette pressure was also seen in the cell-attached configuration
during superfusion with hypotonic medium.

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Fig. 8.
Activation of SA channels in a cell-attached patch of a Reissner's
membrane epithelial cell by pressure and after cell swelling in a
hyposmotic solution. Pipette solution contained 145 mmol/l KCl, control
bath solution contained 140 mmol/l NaCl (~300 mosmol/l), and
hypotonic bath solution contained 100 mmol/l NaCl (~200 mosmol/l).
Vc was 60
mV. A: stretch activation of 11 channels in a cell-attached patch by application of negative pressure
to the pipette. Top: current
recording; bottom: negative pressure
on same time scale. Returning the pressure to zero closed nearly all
channels. O1, O2, O3, and O4 ..., indicate opened levels.
B:
top shows the continuous trace of same
cell-attached patch, before (left open
horizontal bar), during (solid horizontal bar), and after
(right open horizontal bar)
superfusion with hyposmotic solution.
Bottom: time course of
Po for number of
channels in the patch
(NPo) before
(left open bars), during exposure to
hyposmotic solution (crosshatched bars), and after washout
(right open bars). Superfusion with
control solution did not increase
NPo, whereas
hyposmotic solution caused openings of SA channels after a latency of
~30 s. Effect was reversible after washout with the control solution
after a latency period of 30 s. Time scales of the traces and
histograms are identical.
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Channel kinetics.
The high density of the SA channels made kinetic analysis impossible in
most of the patches. Only 8 patches of the 117 tested had only one
level and could be used to analyze the open and closed time
distribution. The influences of voltage, mechanical stretch, and
osmotic pressure on the kinetics of the channel were determined. The
kinetics of channel opening were best described by two time constants
for the open state and the closed state (Table
1). Depolarization mainly decreased the
long closed time constant and increased the short and the long open
time constants (see Fig. 9). This suggests
that the voltage sensitivity is produced by reducing the interval
between bursts of activity and modifies the intraburst kinetics by
stabilizing the open state and reducing the flickering activity. In
contrast, pressure and osmotic swelling modified differently the
distribution of dwell times. The short and the long closed time
constants were decreased, and the long open time constant was increased
(see Fig. 10). This suggests that pressure and osmotic swelling act by destabilizing the closed state and
stabilizing the open state between bursts, without altering the
flickering activity. The differences in the kinetics for voltage dependency and stretch suggest different underlying mechanisms. The
identical changes caused by mechanical and osmotic stretch suggest that
the same mechanisms are involved. The effect of mechanical stretch on
both closed and open time constants has also been reported recently by
Mienville et al. (15). Although the activation by osmotic swelling was
reversible with washout of the hypotonic solution, the modification of
the closed state did not seem to be immediately reversible. Table 1
shows that the effect of washout was mainly on the long open state
constant, acting by decreasing the length of the long openings.
View this table:
[in this window]
[in a new window]
|
Table 1.
Open probability and time constants for SA channels in response to
depolarization, pressure gradient, and hyposmotic stress
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|

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|
Fig. 9.
Kinetic properties of the SA channel: activation by depolarization.
A and
B: open and closed time histograms
were obtained from 1 inside-out patch containing only 1 SA channel at
negative ( 60 mV; A) and
positive (+60 mV; B) membrane
potentials. Intrapipette pressure was 20 mmHg, and duration of each
recording was 60-90 s. Data were low-pass filtered at 1 kHz and
digitized at 2 kHz, and idealized recordings were constructed. Curves
were best fitted by the sum of 2 exponentials to the dwell-time data by
a least-squares method. Events shorter than 1 ms were excluded from the
fit. 1, Fast time constant.
2, slow time constant.
Recordings at bottom of
A and
B are samples from the same patch.
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|

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Fig. 10.
Kinetic properties of the SA channel: activation by pressure.
A and
B: open and closed time histograms
were obtained from 1 inside-out patch containing only 1 SA channel
without intrapipette pressure (A)
and with a negative pressure of 50 mmHg
(B).
Vm was held to
60 mV, and duration of each recording was 60-90 s. Data
were low-pass filtered at 1 kHz and digitized at 2 kHz, and idealized
recordings were constructed. Curves were best fitted by the sum of 2 exponentials to the dwell-time data by a least-squares method. Events
shorter than 1 ms were excluded from the fit. Recordings at
bottom of
A and
B are samples from the same patch.
|
|
 |
DISCUSSION |
This study shows the predominance of SA channels in the apical membrane
of epithelial cells of RM. These channels have some biophysical
properties in common with the family of
Ca2+-activated nonselective
cationic channels, the CAN channels, found in many epithelial tissues
(for a review, see Ref. 18), such as a 20- to 25-pS conductance and an
equal permeability to K+ and
Na+, and are thus difficult to
distinguish under standard conditions (Ringer saline solutions with a
normal extracellular Ca2+
concentration). However, SA channels in the RM epithelial cells differ
strongly from the typical CAN channels by their sensitivity to
pressure, their high voltage dependence, their particular gating kinetics with bursts of flickering, their insensitivity to internal Ca2+, ATP, and fenamates, and
their high permeability to divalent cations. Although most of the CAN
channels reported in the literature were insensitive to pressure or
blockers of SA channels such as Gd3+ and amiloride and poorly
permeable to divalent cations, these distinctives properties seem to be
less important in other tissues. The CAN channels in cultured
epithelial cells of the inner medullary collecting duct (17) and in
intestinal cells (19) are blocked by internal
Gd3+, and those in fetal lung
cells are blocked by external amiloride (25). However, the external
blockade by low concentration of Gd3+ seems to be more specific to
SA channels and has not yet been described in CAN channels but has been
described for Ca2+-blocked
nonselective cation channels (14). Light et al. (11) found that the
25-pS amiloride-sensitive channels in cultured epithelial cells of the
inner medullary collecting duct were activated by internal
Ca2+ in some patches and were
insensitive to Ca2+ in others.
These differences between tissues and species suggest the presence of
two families of channels with common properties, the SA channels and
the CAN channels, that could belong to the same superfamily of
channels, the "degenerins," as suggested by Canessa et al. (2)
and Lingueglia et al. (12) for epithelial amiloride-sensitive
Na+ channels. The RM may contain
SA channels with pure properties, whereas other tissues could have
channels with mixed properties.
Characteristics of SA channels.
Other types of mechanosensitive channels have been described in
neurosensory cells of the inner ear, such as the hair bundle of
vestibular hair cells (5) and the lateral wall of cochlear hair cells
(3, 8). These other channels are probably directly involved in
mechanotransduction processes (7) and differ from the SA channels
described in this study by having higher unit conductance (50-100
pS). However, they are also blocked by low concentrations of external
Gd3+ and amiloride. Although
amiloride sensitivity was not studied here, the SA channels could
contribute to the high density of amiloride-sensitive proteins recently
found on the endolymphatic side of the RM (16).
The conductive properties of the SA channels of the RM, including the
conductance and permeability ratios, resemble those of cardiac atrial
cells (9), cerebral capillaries (20), and the porcine endocardial
endothelium (6). The threshold of activation near 15 mmHg is in the
range of sensitivity to pressure found in most of the cell types. Our
results indicate that a steeper gradient of osmotic pressure is needed
to activate the channel in situ than is suggested by the low
intrapipette pressure required to open SA channels in the cell-attached
configuration. The mean Po was increased
from 0.02 ± 0.005 to 0.13 ± 0.06 in the patches responding to a
difference of 80-100 mosmol/l of osmotic pressure. The same
increase in Po
was obtained with a pressure as low as 20 mmHg applied directly to the
interior of the pipette (see Fig. 3B). A similar difference of
magnitude has been reported in other studies, an increase of the cell
volume near 50% being often necessary to open SA channels (for a
review, see Ref. 21). In our study, mechanically or osmotically induced
stretch exerted similar effects on the open and closed time constants,
suggesting a common underlying mechanism of gating.
Putative role of SA channels in regulation of endolymph pressure and
ion composition.
One of the main properties of the RM is its tightness to the
electrolytes contained in the endolymph, mainly
K+ and water. However, the RM is
not a passive, impermeant membrane, but a structure capable of ion
transport. The transport mechanisms probably lie in the tight monolayer
of confluent epithelial cells facing the endolymph, rather than in the
mesenchymal cells, as they are not confluent and form a leaky layer.
The finding of SA channels in the RM is not surprising, since they have
been found in many other epithelia; the particular structure of this epithelium, the apparent absence of these channels from mesenchymal cells, and their high density suggest that these channels play an
important functional role. This role is certainly related to the
putative functions of this tissue, particularly the regulation of
endolymphatic pressure and the homeostasis of
K+ in the endolymph.
One of the most important questions is whether the range of pressure
needed to activate SA channels is compatible with a physiological or a
pathological role for them. Several controversial studies have
attempted to measure the endolymph pressure during experimental endolymphatic hydrops. Kitahara et al. (10) found that a minimal pressure of 40 mmHg was necessary to disrupt the RM and that 20 mmHg
was needed to distend the membrane. This pressure is near the range
required for activation of SA channels, but it seems difficult to
compare the pressure gradient in the patch pipette and the tension
applied in vivo to the whole RM. The activation of SA channels could be
involved in the electrochemical changes in the endolymph that take
place during the development of clinical (26) and experimental (22)
hydrops. These experimental models have shown that the endolymph
concentrations of K+ and
Cl
change during the
development of endolymphatic hydrops in two stages. In the first stage,
there is a gradual distension of the RM with no measurable increase in
the endolymph pressure and no change in ionic concentrations or
osmolality. The activity of SA channels is probably below the threshold
of activation during this period. The second period is marked by
electrochemical changes and an increase in hydrostatic pressure that
could activate SA channels. These channels are permeable to
K+, the major ion in the
endolymph, and thus could contribute to the leak of
K+ that occurs during hydrops.
They are also permeable to Ca2+
and could in turn activate
Ca2+-dependent channels like the
CAN channels. This type of interaction between SA channels and
high-conductance Ca2+-activated
K+ channels (maxi-K+
channels) has recently been described in other tissues (6) but was not
observed with CAN channels in our experiments. The increase in internal
Ca2+ could also act as a
transducer of Ca2+-dependent
internal biochemical processes, like the activation of enzymes. This
cascade could explain the morphological alterations, thickening, or
rupture of the epithelium of the RM that often occurs at this stage
(31). The fact that SA channels are regulated by both mechanical
pressure and osmotic changes could also explain the empirical efficacy
of treatment with osmotic substances, such as mannitol or glycerol, of
humans suffering from endolymphatic hydrops.
In conclusion, this study demonstrates that the patch-clamp technique
is suitable for studying ion channels in freshly dissected out RM
epithelial cells. The finding of an SA channel in high density in this
tissue, which undergoes physiological and pathological changes in
pressure, seems of great significance. Stretch might be the main
regulator of these channels, but further experiments are required to
investigate other possible regulators.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Evelyne Ferrary and Dr. Jacques Teulon for reviewing the
manuscript and Dr. Owen Parkes for checking the English text.
 |
FOOTNOTES |
This work was supported by National Science Council of Taiwan Grant
NSC-85-2-331-B002-230.
A preliminary characterization of the channel studied here was included
in a previous report (T.-H. Yeh, M. C. Tsai, S.-Y. Lee, M.-M. Hsu, and
P. Tran Ba Huy. Hear. Res. 109:1-10, 1997).
Address for reprint requests: T. Van Den Abbeele, Laboratoire
d'Otologie Expérimentale, Faculté Lariboisiére, 10 Ave. de Verdun, 75010 Paris, France.
Received 31 March 1997; accepted in final form 15 September 1997.
 |
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