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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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+ approx  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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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 MOmega ) 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
<IT>P</IT><SUB>o</SUB> = <IT>P</IT>(0) + <FR><NU>1</NU><DE>1 + <IT>e</IT><SUP>−(<IT>V</IT><SUB>m</SUB>− <IT>V</IT><SUB>50</SUB>)/<IT>d</IT></SUP></DE></FR>
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
<IT>P</IT><SUB>o</SUB> = <IT>P</IT>(0) + <FR><NU><IT>P</IT><SUB>max</SUB></NU><DE>1 + <IT>e</IT><SUP>−(P−P<SUB>50</SUB>)/<IT>d</IT></SUP></DE></FR>
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 · <IT>e</IT><SUP>−<IT>t</IT>/&tgr;<SUB>1</SUB></SUP> + B · <IT>e</IT><SUP>−<IT>t</IT>/&tgr;<SUB>2</SUB></SUP>, where N is the number of events, A and B are the coefficients, and tau 1 and tau 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(beta -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).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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). bullet , Po values (means ± SE) of 8 cell-attached patches. open circle , 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.

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. bullet , Current values (means ± SE) of 24 cell-attached patches. triangle , 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.

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 (open circle , n = 7) or 90 mmol/l CaCl2 (bullet , 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). open circle , Po values (means ± SE) of 7 excised inside-out patches with 90 mmol/l BaCl2 inside the pipette. bullet , 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.

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. bullet , Po (means ± SE) of 5 inside-out patches when the bath contained 1 mmol/l CaCl2. open circle , 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. bullet , Po (means ± SE) of 4 inside-out patches when the bath contained 1 mmol/l flufenamic acid without CaCl2. open circle , 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).

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. bullet , Po (means ± SE) of 7 inside-out patches. open circle , 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.

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.

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.

                              
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Table 1.   Open probability and time constants for SA channels in response to depolarization, pressure gradient, and hyposmotic stress


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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. tau 1, Fast time constant. tau 2, slow time constant. Recordings at bottom of A and B are samples from the same patch.


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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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 274(3):C566-C576
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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