Modulation of Outer Hair Cell Electromotility by Cochlear Supporting Cells and Gap Junctions

Outer hair cell (OHC) or prestin-based electromotility is an active cochlear amplifier in the mammalian inner ear that can increase hearing sensitivity and frequency selectivity. In situ, Deiters supporting cells are well-coupled by gap junctions and constrain OHCs standing on the basilar membrane. Here, we report that both electrical and mechanical stimulations in Deiters cells (DCs) can modulate OHC electromotility. There was no direct electrical conductance between the DCs and the OHCs. However, depolarization in DCs reduced OHC electromotility associated nonlinear capacitance (NLC) and distortion products. Increase in the turgor pressure of DCs also shifted OHC NLC to the negative voltage direction. Destruction of the cytoskeleton in DCs or dissociation of the mechanical-coupling between DCs and OHCs abolished these effects, indicating the modulation through the cytoskeleton activation and DC-OHC mechanical coupling rather than via electric field potentials. We also found that changes in gap junctional coupling between DCs induced large membrane potential and current changes in the DCs and shifted OHC NLC. Uncoupling of gap junctions between DCs shifted NLC to the negative direction. These data indicate that DCs not only provide a physical scaffold to support OHCs but also can directly modulate OHC electromotility through the DC-OHC mechanical coupling. Our findings reveal a new mechanism of cochlear supporting cells and gap junctional coupling to modulate OHC electromotility and eventually hearing sensitivity in the inner ear.     

Electrical Properties of Structural Components of the Crystalline Lens

The electrical properties of the crystalline lens of the frog eye are measured with stochastic currents applied with a microelectrode near the center of the preparation and potential recorded just under the surface. The stochastic signals are decomposed by Fourier analysis into sinusoidal components, and the impedance is determined from the ratio of mean cross power to input power. The data are fit by an electrical model that includes two paths for current flow: one through the cytoplasm, gap junctions, and outer membrane; the other through inner membranes and the extracellular space between lens fibers. The electrical properties of the structures of the lens which appear as circuit components in the model are determined by the fit to the data. The resistivity of the extracellular space within the lens is comparable to the resistivity of Ringer. The outer membrane has a normal resistance of 5 kohm · cm² but large capacitance of 10 μF/cm², probably because it represents the properties of several layers of fibers. The inner membranes have properties reminiscent of artificial lipid bilayers: they have high membrane resistance, 2.2 megohm · cm², and low specific capacitance, 0.8 μF/cm². There is so much membrane within the lens, however, that the sum of the current flow across all the inner membranes is comparable to that across the outer surface.     

Double whole-cell patch-clamp characterization of gap junctional channels in isolated insect epidermal cell pairs

Double whole-cell patch-clamp methods were used to characterize Junctional membrane conductances in epidermal cell pairs isolated from the prepupal integument of the flour beetle, Tenebrio molitor. The mean initial Junctional conductance in 267 cell pairs was 9.5 ± 1.0 nS (range 0–95 nS). Well-coupled cell pairs uncoupled spontaneously with a half-time of 7.6 min. Adding 5 mM ATP to the pipette solution stabilized coupling with less than a 50% drop occurring after 30 min. Nonjunctional membrane potential was the major determinant of Junctional conductance with transjunctional potential playing a minor role. Junctional conductance approached 0 pA at nonjunctional membrane potentials greater than 0 mV and increased with hyperpolarization. The voltage at half-maximal conductance was –26 mV. The time course of the reversible changes in Junctional conductance were slow (30 sec) with time-dependent decay occurring faster and recovery occurring slower with increasing depolarization. Single gap Junctional channel activity was recorded in uncoupling cell pairs and in poorly coupled ATP-stabilized cell pairs. One main single channel conductance was observed in each cell pair. The mean single channel conductances from all cell pairs in this study ranged from 197–347 pS (mean 248 pS). Single channel conductance was linear over the ±60 mV transjunctional voltage range tested. A broad range of subconductance states of the main state representing 5% of the total open time of measurable main state events was observed. Single channel activity was strongly dependent on the nonjunctional membrane potential, increasing with hyperpolarization.We gratefully acknowledge the helpful advice of Dr. Stephen Sims. This work was supported by NSERC of Canada grant No. A6797 to S.C. D.C. was supported by an NSERC scholarship for part of this work.     

ATP activates P2X receptors to mediate gap junctional coupling in the cochlea

ATP is an important extracellular signaling molecule and can activate both ionotropic (P2X) and metabotropic purinergic (P2Y) receptors to influence cellular function in many aspects. Gap junction is an intercellular channel and plays a critical role in hearing. Here, we report that stimulation of ATP reduced gap junctional coupling between cochlear supporting cells. This uncoupling effect could be evoked by nanomolar physiological levels of ATP. A P2X receptor agonist benzoylbenzoyl-ATP (BzATP) but not a P2Y receptor agonist UTP stimulated this uncoupling effect. Application of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or oxidized ATP (oATP, 0.1 mM) eliminated this uncoupling effect. We further found that ATP activated P2X receptors in the cochlear supporting cells allowing Ca²⁺ influxing, thereby increasing intracellular Ca²⁺ concentration to mediate gap junctions. These data suggest that ATP can mediate cochlear gap junctions at the physiological level by the activation of P2X receptors rather than P2Y receptors. This P2X receptor-mediated purinergic control on the cochlear gap junctions may play an important role in the regulation of K⁺-recycling for ionic homeostasis in the cochlea and the reduction of hearing sensitivity under noise stress for protection.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Biophysics of the inhibition of the growth of maize roots by lowered temperature

Roots of hydroponically grown maize (Zea mays cv LG11) have a greatly reduced growth rate at 5°C (0.02 millimeters per hour) compared with those at 20°C (1.2 millimeters per hour). Various physical parameters of roots growing at each temperature were compared. Turgor pressure of cells in the elongation zone increased from 0.59 ± 0.05 megapascal at 20°C to 0.82 ± 0.04 megapascal after 70 hours at 5°C; thus, growth rate was not limited by decreased pressure. On cooling, tissue plasticity (measured by Instron/tensiometer) decreased slowly over 70 hours. On rewarming to 20°C from 5°C, growth rate, turgor pressure, and tissue plasticity all returned concertedly to their original values over a period of days. However, immediately following cooling growth rate dropped rapidly from 1.8 to 0.12 millimeters per hour in 30 minutes but turgor pressure and tissue Instron plasticity remained unchanged. A plot of turgor pressure against growth rate indicated that, following cooling from 30 to 15°C, the in vivo wall extensibility of the tissue was reduced by 75%. Yield threshold was unchanged. Cells whose expansion was arrested in the long-term cold treatment do not resume growth. Root growth recovers by the expansion of cells newly produced by the meristem. Cessation of extension growth is an effect on the individual expanding cell. Growth recovery is not a reverse of this effect but requires the generation of fresh cells.     

Development changes in regulation of embryonic chick heart gap junctions

Summary Embryonic chick myocyte pairs were isolated from ventricular tissue of 4-day, 14-day, and 18-day heart for the purpose of examining the relationship between macroscopic junctional conductance and transjunctional voltage during cardiac development. The double whole-cell patch-clamp technique was employed to directly measure junctional conductance over a transjunctional voltage range of ±100 mV. At all ages, the instantaneous junctional current (or conductance=current/voltage) varied linearly with respect to transjunctional voltage. This initial response was followed by a time- and voltage-dependent decline in junctional current to new steady-state values. For every experiment, the steady-state junctional conductance was normalized to the instantaneous value obtained at each potential and the data was pooled according to developmental age. The mean steadystate junctional conductance-voltage relationship for each age group was fit using a two-state Boltzmann distribution described previously for other voltage-dependent gap junctions. From this model, it was revealed that half-inactivation voltage for the transjunctional voltage-sensitive conductance shifted towards larger potentials by 10 mV, the equivalent gating charge increased by approximately 1 electron, and the minimal voltage-insensitive conductance exactly doubled (increased from 18 to 36%) between 4 and 18 days of development. Decay time constants were similar at all ages examined as rate increased with increasing transjunctional potential. This data provides the first direct experimental evidence for developmental changes in the regulation of intercellular communication within a given tissue. This information is consistent with the hypothesis that developmental expression of multiple gap junction proteins (connexins) may confer different regulatory mechanisms on intercellular communication pathways within a given cell or tissue.     

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".     

Equilibrium properties of a voltage-dependent junctional conductance

The conductance of junctions between amphibian blastomeres is strongly voltage dependent. Isolated pairs of blastomeres from embryos of Ambystoma mexicanum, Xenopus laevis, and Rana pipiens were voltage clamped, and junctional current was measured during transjunctional voltage steps. The steady-state junctional conductance decreases as a steep function of transjunctional voltage of either polarity. A voltage-insensitive conductance less than 5% of the maximum remains at large transjunctional voltages. Equal transjunctional voltages of opposite polarities produce equal conductance changes. The conductance is half maximal at a transjunctional voltage of approximately 15 mV. The junctional conductance is insensitive to the potential between the inside and outside of the cells. The changes in steady-state junctional conductance may be accurately modeled for voltages of each polarity as arising from a reversible two-state system in which voltage linearly affects the energy difference between states. The voltage sensitivity can be accounted for by the movement of about six electron charges through the transjunctional voltage. The changes in junctional conductance are not consistent with a current-controlled or ionic accumulation mechanism. We propose that the intramembrane particles that comprise gap junctions in early amphibian embryos are voltage-sensitive channels.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.

The electrical properties of gap junctions in cell pairs are usually studied by means of the dual voltage clamp method. The voltage across the junctional channels, however, cannot be controlled adequately due to an artificial resistance and a natural resistance, both connected in series with the gap junction. The access resistances to the cell interior of the recording pipettes make up the artificial resistance. The natural resistance consists of the cytoplasmic access resistances to the tightly packed gap junction channels in both cells. A mathematical model was constructed to calculate the actual voltage across each gap junction channel. The stochastic open-close kinetics of the individual channels were incorporated into this model. It is concluded that even in the ideal case of complete compensation of pipette series resistance, the number of channels comprised in the gap junction may be largely underestimated. Furthermore, normalized steady-state junctional conductance may be largely overestimated, so that transjunctional voltage dependence is easily masked. The model is used to discuss conclusions drawn from dual voltage clamp experiments and offers alternative explanations for various experimental observations.     

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.     

A Highlights from MBoC Selection: Inhibition of cell membrane ingression at the division site by cell walls in fission yeast

Eukaryotic cells assemble actomyosin rings during cytokinesis to function as force-generating machines to drive membrane invagination and to counteract the intracellular pressure and the cell surface tension. How the extracellular matrix affects actomyosin ring contraction has not been fully explored. While studying the Schizosaccharomyces pombe 1,3-β-glucan-synthase mutant cps1-191, which is defective in division septum synthesis and arrests with a stable actomyosin ring, we found that weakening of the extracellular glycan matrix caused the generated spheroplasts to divide under the nonpermissive condition. This nonmedial slow division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. Interestingly, the high intracellular turgor pressure appears to play a minimal role in inhibiting ring contraction in the absence of cell wall remodeling in cps1-191 mutants, as decreasing the turgor pressure alone did not enable spheroplast division. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.     

Pressure regulation of the electrical properties of growing Arabidopsis thaliana L. root hairs.

Actively growing Arabidopsis thaliana L. (Columbia wild type) root hairs were used to examine the interplay between cell turgor pressure and electrical properties of the cell: membrane potential, conductance, cell-to-cell coupling, and input resistance. Pressure was directly modulated using a pressure probe or indirectly by changing the extracellular osmolarity. Direct modulation of pressure in the range of 0 to about 15 x 10(5) Pa (normal turgor pressure was 6.8 +/- 2.0 x 10(5) Pa, n = 29) did not affect the membrane potential, conductance, coupling, or input resistance. Indirect modulation of turgor pressure by adding (hyperosmotic) or removing (hypo-osmotic) 200 mM mannitol/sorbitol affected the potential and conductance but not cell-to-cell coupling. Hypo-osmotic treatment depolarized the potential about 40 mV from an initial potential of about -190 mV and increased membrane conductance, consistent with an increase in anion efflux from the cell. Hyperosmotic treatment hyperpolarized the cell about 25 mV from the same initial potential and decreased conductance, consistent with a decline in cation influx. The results are likely due to the presence of an "osmo-sensor," rather than a "turgor-sensor," regulating the cell's response to osmotic stress.     

Pressure probe technique for measuring water relations of cells in higher plants

A new method is described for continuously measuring cell turgor pressure (P), hydraulic conductivity (L_p), and volumetric elastic modulus (ε) in higher plant cells, using a pressure probe. This technique permits volume changes, ΔV, and turgor pressure changes, ΔP, to be determined with an accuracy of 10⁻⁵ to 10⁻⁶ μl and 3 to 5·10⁻² bar, respectively.

The main principle of the new method is the same as the pressure probe developed by Zimmermann and Steudle in which pressure is transmitted to a pressure transducer by means of an oil-filled capillary introduced into the cell. In order to use the pressure probe for small tissue cells, the effective compressible volume of the apparatus has to be sufficiently small in comparison to the volume of the cell itself. This is achieved by accurately fixing the oil/cell sap boundary in the very tip of the microcapillary by means of an electronic feedback mechanism, so that the effective volume of the apparatus is reduced to about 2 to 10% of the cell volume. In this way also, errors arising from compressibility of the apparatus and temperature fluctuations can be excluded.

Measurements on tissues cells of Capsicum annuum fruits yield ε values of 2 to 25 bar. Furthermore, ε can be shown to be a function of both cell turgor pressure and cell volume; ε increases with increasing turgor pressure and is higher in larger cells.

     

Cell turgor changes associated with ripening in tomato pericarp tissue

The pressure microprobe was used to determine whether the turgor pressure in tomato (Lycopersicon esculentum Mill., variety “Castelmart”) pericarp cells changed during fruit ripening. The turgor pressure of cells located 200 to 500 micrometers below the fruit epidermis was uniform within the same tissue (typically ± 0.02 megapascals), and the highest turgors observed (<0.2 megapascals) were much less than expected, based on tissue osmotic potential (−0.6 to −0.7 megapascals). These low turgor values may indicate the presence of apoplastic solutes. In both intact fruit and cultured discs of pericarp tissue, a small increase in turgor preceded the onset of ripening, and a decrease in turgor occurred during ripening. Differences in the turgor of individual intact fruit occurred 2 to 4 days before parallel differences in their ripening behavior were apparent, indicating that changes in turgor may reflect physiological changes at the cell level that precede expression of ripening at the tissue level.     

Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.     

Acid–base titration across the membrane system of rat-liver mitochondria: Catalysis by uncouplers

1. Pulsed acid–base titrations of suspensions of rat-liver mitochondria under anaerobic equilibrium conditions show fast and slow titration processes. 2. The fast process is the titration of the outer aqueous phase of the mitochondria, which is continuous with the suspension medium, and the slow process can be identified with the titration of the inner aqueous phase of the mitochondria, which is separated from the outer aqueous phase by the non-aqueous osmotic barrier or M phase of the cristae membrane system. 3. The buffering power of the outer and inner phases have been separately measured over a range of pH values. 4. The rate of titration of the inner aqueous phase under a known protonmotive force across the M phase has been characterized by an effective proton conductance coefficient, which, near pH7 and at 25°, is only 0·45μmho/cm.² of the M-phase membrane. 5. The low effective proton conductance of the M phase will account quantitatively for the observed respiratory control in state 4, assuming that oxidoreduction and phosphorylation are coupled by a circulating proton current as required by the chemi-osmotic hypothesis. 6. The addition of 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) at normal uncoupling concentrations causes a large increase in the effective proton conductance of the M phase of the cristae membrane. 7. The increase of the effective proton conductance of the M phase by 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) will account quantitatively for the short-circuiting effect of the uncoupling agent on the proton current and for the observed rise of the rate of respiration to that characteristic of state 3 or higher.     

Gap Junctions and Cochlear Homeostasis

Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations. H.-B. Zhao, T. Kikuchi, A. Ngezahayo, T. W. White contributed equally to this article     

Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G _j) and cytoplasmic concentration of free Ca²⁺ ([Ca²⁺]_i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G _j varied for different cell pairs. One population exhibited a bell-shape dependence of G _j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca²⁺]_i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca²⁺, blockage of K⁺ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca²⁺ or blockage of K⁺ efflux, formation of whole-cell configuration generated a Ca²⁺ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca²⁺ release from intracellular Ca²⁺ stores, which activates sustained Ca²⁺ influx, K⁺ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.