Tuning in to the amazing outer hair cell: membrane wizardry with a twist and shout.
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Animals↗
Biomedical subjects
J Santos-Sacchi
Publications and source records attributed to J Santos-Sacchi.
Protein- and lipid-reactive agents alter outer hair cell lateral membrane motor charge movement.
Outer hair cells from the mamma*lian cochlea are mechanically active cells that rely on charged voltage sensors within their lateral plasma membrane to gate the integral membrane motor protein, prestin, into one of two area states. Here we use protein and lipid reactive reagents to probe the influence of these bilayer components on motor-induced nonlinear membrane capacitance. Of the protein-reactive reagents tested, cross-linking and sulfhydryl reagents were most effective in altering steady state and time-varying motor activity. Of the lipid-altering agents, chloroform and HePC were most effective. Chloroform, in particular, drastically modified the susceptibility of the motor to prior voltage (initial conditions). Our data suggest that outer hair cell motor activity derives substantially from interactions with its lipid environment.
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Temperature dependence of non-linear capacitance in human embryonic kidney cells transfected with prestin, the outer hair cell motor protein.
The transmembrane motor protein prestin is thought to underlie outer hair cell (OHC) motility. Prestin expressed in non-auditory cells confers OHC-like electrical characteristics to the cell membrane, including the generation of gating-like currents (or non-linear capacitance), whose voltage dependence is susceptible to membrane tension and initial voltage conditions. Here we report that prestin's voltage sensitivity is, like that of the native motor, markedly temperature dependent. Prestin-transfected HEK cells were whole-cell voltage clamped while temperature was varied from 10-35 degrees C. V(pkcm), the voltage at peak capacitance, reversibly and linearly shifted to depolarized levels with increasing temperatures, while peak capacitance also increased, but with significant hysteresis upon recooling. Mathematical modeling suggests that this increase may be due to a charged voltage sensor having a wider range of movement through or larger unit charge within the plasma membrane at higher temperatures.
Anion Transport Proteins↗
Effects of membrane potential and tension on prestin, the outer hair cell lateral membrane motor protein.
1. Under whole-cell voltage clamp, the effects of initial voltage conditions and membrane tension on gating charge and voltage-dependent capacitance were studied in human embryonic kidney cells (TSA201 cell line) transiently transfected with the gene encoding the gerbil protein prestin. Conformational changes in this membrane-bound protein probably provide the molecular basis of the outer hair cell (OHC) voltage-driven mechanical activity, which spans the audio spectrum. 2. Boltzmann characteristics of the charge movement in transfected cells were similar to those reported for OHCs (Q(max) = 0.99 +/- 0.16 pC, z = 0.88 +/- 0.02; n = 5, means +/- S.E.M.). Unlike that of the adult OHC, the voltage at peak capacitance (V(pkcm)) was very negative (-74.7 +/- 3.8 mV). Linear capacitance in transfected cells was 43.7 +/- 13.8 pF and membrane resistance was 458 +/- 123 Mohms. 3. Voltage steps from the holding potential preceding the measurement of capacitance-voltage functions caused a time- and voltage-dependent shift in V(pkcm). For a prepulse to -150 mV, from a holding potential of 0 mV, V(pkcm) shifted 6.4 mV, and was fitted by a single exponential time constant of 45 ms. A higher resolution analysis of this time course was made by measuring the change in capacitance during a fixed voltage step and indicated a double exponential shift (tau(0) = 51.6 ms, tau(1) = 8.5 s) similar to that of the native gerbil OHC. 4. Membrane tension, delivered by increasing pipette pressure, caused a positive shift in V(pkcm). A maximal shift of 7.5 mV was obtained with 2 kPa of pressure. The effect was reversible. 5. Our results show that the sensitivity of prestin to initial voltage and membrane tension, though present, is less than that observed in adult OHCs. It remains possible that some other interacting molecular species within the lateral plasma membrane of the native OHC amplifies the effect of tension and prior voltage on prestin's activity.
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Non-uniform mapping of stress-induced, motility-related charge movement in the outer hair cell plasma membrane.
There is a growing consensus that outer hair cell (OHC) electromotility underlies the mammalian cochlear amplifier. This voltage-dependent motility is mirrored by a gating current, which along with motility can be altered by tension applied to the cell's plasma membrane. We used localized tension application along the length of the OHC to induce gating currents from membrane microdomains; with this information we mapped the distribution of the OHC's sensitivity to membrane stress before and after disrupting the cytoskeleton with intracellular Pronase. Mechanically induced gating currents, which were susceptible to salicylate, lanthanides and turgor pressure, evidenced a bell-shaped distribution that was restricted to the lateral membrane where the electromotile response resides. After Pronase treatment, gating currents remained intact and restricted. These results confirm that the molecular motors are intrinsically and bi-directionally susceptible to voltage and tension, and provide evidence for limited mobility of OHC motors within the cell's lateral membrane.
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Furosemide alters nonlinear capacitance in isolated outer hair cells.
The outer hair cell (OHC) from the organ of Corti plays a crucial role in hearing through its unique voltage-dependent mechanical responses. Furosemide, one of the loop diuretics, disrupts normal cochlear function. Here we report on direct effects of furosemide on OHC motility-related, voltage-dependent capacitance using the whole-cell patch-clamp technique. Extracellularly applied furosemide reversibly shifted the voltage at peak capacitance (V(pkC(m))) to positive levels. The shift, whose maximum approached 90 mV, evidenced a Hill coefficient of 1.5 and K(1/2) of 10 mM. Changes in the magnitude of nonlinear capacitance were not fully reversible. While it is clear that the overwhelming effect of furosemide on hearing results via its effects on the endolymphatic potential, the present results indicate that furosemide directly alters OHC motility and may, in part, contribute to sensory dysfunction.
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Voltage gating of gap junctions in cochlear supporting cells: evidence for nonhomotypic channels.
The organ of Corti has been found to have multiple gap junction subunits, connexins, which are localized solely in nonsensory supporting cells. Connexin mutations can induce sensorineural deafness. However, the characteristics and functions of inner ear gap junctions are not well known. In the present study, the voltage-dependence of gap junctional conductance (G(j)) in cochlear supporting cells was examined by the double voltage clamp technique. Multiple types of asymmetric voltage dependencies were found for both nonjunctional membrane voltage (V(m)) and transjunctional (V(j)) voltage. Responses for each type of voltage dependence were categorized into four groups. The first two groups showed rectification that was polarity dependent. The third group exhibited rectification with either voltage polarity, i.e., these cells possessed a bell-shaped G(j)-V(j) or G(j)-V(m) function. The rectification due to V(j) had fast and slow components. On the other hand, V(m)-dependent gating was fast (<5 msec), but stable. Finally, a group was found that evidenced no voltage dependence, although the absence of V(j) dependence did not preclude V(m) dependence and vice versa. In fact, for all groups V(j) sensitivity could be independent of V(m) sensitivity. The data show that most gap junctional channels in the inner ear have asymmetric voltage gating, which is indicative of heterogeneous coupling and may result from heterotypic channels or possibly heteromeric configurations. This heterogeneous coupling implies that single connexin gene mutations may affect the normal physiological function of gap junctions that are not limited to homotypic configurations.
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Nitric oxide uncouples gap junctions of supporting Deiters cells from Corti's organ.
Supporting cells of Corti's organ are electrically coupled via gap junctions. They probably serve to maintain the unique cochlear environment that is required for normal sensory function. In this study we used input capacitance measurements under whole-cell voltage-clamp conditions to evaluate the effects of nitric oxide on gap junctional communication between pairs of isolated supporting Deiters cells. We show that the nitric oxide (NO) donor sodium nitroprusside causes the uncoupling of Deiters cells, and that an NO synthase inhibitor blocks the effect. The cGMP analogue 8-bromo-cGMP also uncouples Deiters cells. With either treatment, the input capacitance of pairs of Deiters cells drops to single-cell levels within minutes of application, indicative of electrical uncoupling. We surmise that the NO/cGMP pathway may serve to modulate normal cochlear homeostasis and possibly plays a role in ototoxic mechanisms.
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Cell coupling in Corti's organ.
The mammalian organ of Corti is responsible for the initial analysis of sound; injury leads to hearing loss. During the last two decades, the characteristics of cellular coupling in this specialized epithelium have been studied. In this review, data on both electrical and mechanical coupling are covered. While electrical coupling likely contributes to homeostasis in the organ, this concept is far from proven.
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Distortion component analysis of outer hair cell motility-related gating charge.
The underlying Boltzmann characteristics of motility-related gating currents of the outer hair cell (OHC) are predicted to generate distortion components in response to sinusoidal transmembrane voltages. We studied this distortion since it reflects the mechanical activity of the cell that may contribute to peripheral auditory system distortion. Distortion components in the OHC electrical response were analyzed using the whole-cell voltage clamp technique, under conditions where ionic conductances were blocked. Single or double-sinusoidal transmembrane voltage stimulation was delivered at various holding voltages, and distortion components of the current responses were detected by Fourier analysis. Current response magnitude and phase of each distortion component as a function of membrane potential were compared with characteristics of the voltage-dependent capacitance, obtained by voltage stair-step transient analysis or dual-frequency admittance analysis. The sum distortion was most prominent among the distortion components at all holding voltages. Notches in the sum (f1+f2), difference (f2-f1) and second harmonic (2f) components occur at the voltage where peak voltage-dependent capacitance resides (VpkCm). Rapid phase reversals also occurred at VpkCm, but phase remained fairly stable at more depolarized and hyperpolarized potentials. Thus, it is possible to extract Boltzmann parameters of the motility-related charge movement from these distortion components. In fact, we have developed a technique to follow changes in the voltage dependence of OHC motility and charge movement by tracking the voltage at phase reversal of the f2-f1 product. When intracellular turgor pressure was changed, VpkCm and distortion notch voltages shifted in the same direction. These data have important implications for understanding cochlear nonlinearity, and more generally, indicate the usefulness of distortion analysis to study displacement currents.
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Auditory collusion and a coupled couple of outer hair cells.
The discrepancies between measured frequency responses of the basilar membrane in the inner ear and the frequency tuning found in psychophysical experiments led to Bekesy's idea of lateral inhibition in the auditory nervous system. We now know that basilar membrane tuning can account for neural tuning, and that sharpening of the passive travelling wave depends on the mechanical activity of outer hair cells (OHCs)3, but the mechanism by which OHCs enhance tuning remains unclear. OHCs generate voltage-dependent length changes at acoustic rates, which deform the cochlear partition. Here we use an electrical correlate of OHC mechanical activity, the motility-related gating current, to investigate mechano-electrical interactions among adjacent OHCs. We show that the motility caused by voltage stimulation of one cell in a group evokes gating currents in adjacent OHCs. The resulting polarization in adjacent cells is opposite to that within the stimulated cell, which may be indicative of lateral inhibition. Also such interactions promote distortion and suppression in the electrical and, consequently, the mechanical activity of OHCs. Lateral interactions may provide a basis for enhanced frequency selectivity in the basilar membrane of mammals.
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Effects of lipophilic ions on outer hair cell membrane capacitance and motility.
The outer hair cell (OHC) from the mammalian organ of Corti possesses a bell-shaped voltage-dependent capacitance function. The nonlinear capacitance reflects the activity of membrane bound voltage sensors associated with membrane motors that control OHC length. We have studied the effects of the lipophilic ions, tetraphenylborate (TPB-) and tetraphenylphosphonium (TPP+), on nonlinear capacitance and motility of isolated guinea-pig OHCs. Effects on supporting cells were also investigated. TPB- produced an increase in the peak capacitance (Cmpk) and shifted the voltage at peak capacitance (VpkCm) to hyperpolarized levels. Washout reversed the effects. Perfusion of 0.4 micrometer TPB- caused an average increase in Cmpk of 16.3 pF and VpkCm shift of 13.6 mV. TPP+, on the other hand, only shifted VpkCm in the positive direction, with no change in Cmpk. The contributions from native OHC and TPB--induced capacitance were dissected by a double Boltzmann fitting paradigm, and by blocking native OHC capacitance. While mechanical response studies indicate little effect of TPB- on the motility of OHCs which were in normal condition or treated with salicylate or gadolinium, the voltage at maximum mechanical gain (VdeltaLmax) was shifted in correspondence with native VpkCm, and both changed in a concentration-dependent manner. Both TPB--induced changes in Cmpk and VpkCm were affected by voltage prepulses and intracellular turgor pressure. TPB- induced a voltage-dependent capacitance in supporting cells whose characteristics were similar to those of the OHC, but no indication of mechanical responses was noted. Our results indicate that OHC mechanical responses are not simply related to quantity of nonspecific nonlinear charge moved within the membrane, but to the effects of motility voltage-sensor charge movement functionally coupled to a mechanical effector.
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Density of motility-related charge in the outer hair cell of the guinea pig is inversely related to best frequency.
Whole cell voltage clamp and freeze fracture were used to study the electrophysiological and ultrastructural correlates of the outer hair cell (OHC) lateral membrane molecular motors. We find that specific voltage-dependent capacitance, which derives from motility-related charge movement, increases as cell length decreases. This increasing non-linear charge density predicts a corresponding increase in sensor-motor density. However, while OHC lateral membrane particle density increases, a quantitative correspondence is absent. Thus, the presumed equivalence of particle and motor is questionable. The data more importantly indicate that whereas the voltage driving OHC motility, i.e. the receptor potential, may decrease with frequency due to the OHC's low-pass membrane filter, the electrical energy (Q x V) supplied to the lateral membrane will tend to remain stable. This conservation of energy delivery is likely crucial for the function of the cochlear amplifier at high frequencies.
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Effects of membrane potential on the voltage dependence of motility-related charge in outer hair cells of the guinea-pig.
1. Isolated outer hair cells (OHCs) from the guinea-pig were whole-cell voltage clamped to study the influence of initial voltage on the voltage dependence of motility-related gating current or, equivalently, on the voltage dependence of membrane capacitance. 2. Prepulse delivery caused changes in the magnitude of motility-related gating currents, which are due predominantly to shifts in the voltage at peak capacitance (VpkCmm). Depolarization shifts VpkCm in the hyperpolarizing direction, and hyperpolarization does the opposite. The mean shift between -120 and +40 mV prepulse states with long-term holding potentials (> 2 min) at -80 mV was 14. 67 +/- 0.95 mV (n = 10; mean +/- s.e.m.). 3. The effect of initial membrane potential is sigmoidal, with a voltage dependence of 23 mV per e-fold change in VpkCm, and maximum slope within the physiological range of OHC resting potentials. This indicates that the cell is poised to respond maximally to changes in resting potential. 4. The kinetics of prepulse effects are slow compared with motility-related gating current kinetics. High-resolution measurement of membrane capacitance (Cm) using two voltage sinusoids indicates that shifts in VpkCm induce Cm changes with time courses fitted by two exponentials (tau0, 0.070 +/- 0.003 s; tau1, 1.28 +/- 0.07 s; A0, 1.54 +/- 0.13 pF; A1, 1.51 +/- 0.13 pF; means +/- s.e.m. ; n = 22; step from +50 to -80 mV). Recovery of prepulse effects exhibits a similar time course. 5. Prepulse effects are resistant to intracellular enzymatic digestion, to fast intracellular calcium buffers, and to intracellular pressure. Through modelling, we indicate how the effect may be explained by an intrinsic voltage-induced tension generated by the molecular motors residing in the lateral membrane.
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Temperature dependence of outer hair cell nonlinear capacitance.
The temperature dependence of outer hair cell motility-related gating current and capacitance was evaluated under whole-cell voltage clamp. Temperature change caused a shift of these voltage-dependent functions along the voltage axis, with a decrease in temperature causing a negative shift in the voltage at peak capacitance (Vpkcm) of 19.2 mV per 10 degrees C. Gating current kinetics showed only mild temperature dependence, the Q10 being about 1.5. Temperature is speculated to affect outer hair cell (OHC) mechanical gain and frequency response by alterations in lateral membrane viscoelastic properties. Such temperature-dependent effects on the OHC may mediate known temperature effects on in vivo cochlear physiology.
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Effect of membrane tension on gap junctional conductance of supporting cells in Corti's organ.
The effects of turgor pressure-induced membrane tension on junctional coupling of Hensen cell isolates from the inner ear were evaluated by input capacitance or transjunctional conductance measurement techniques. Turgor pressure was altered by changing either pipette pressure or the osmolarities of extracellular solutions. Both positive pipette pressure and extracellular applications of hypotonic solutions, which caused cell size to concomitantly increase, uncoupled the cells as indicated by reduced input capacitance and transjunctional conductance. These changes were, in many cases, reversible and repeatable. Intracellular application of 50 microM H-7, a broad-based protein kinase inhibitor, and 10 mM BAPTA did not block the uncoupling effect of positive turgor pressure on inner ear gap junctions. The transjunctional conductance at a holding potential of -80 mV was 53.6 +/- 5.8 nS (mean +/- SEM, n = 9) and decreased approximately 40% at a turgor pressure of 1.41 +/- 0.05 kPa. Considering the coincident kinetics of cell deformation and uncoupling, we speculate that mechanical forces work directly on gap junctions of the inner ear. These results suggest that pathologies that induce imbalances in cochlear osmotic pressure regulation may compromise normal cochlear homeostasis.
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Mapping the distribution of outer hair cell voltage-dependent conductances by electrical amputation.
The mammalian outer hair cell (OHC) functions not only as sensory receptor, but also as mechanical effector; this unique union is believed to enhance our ability to discriminate among acoustic frequencies, especially in the kilohertz range. An electrical technique designed to isolate restricted portions of the plasma membrane was used to map the distribution of voltage-dependent conductances along the cylindrical extent of the cell. We show that three voltage-dependent currents, outward K, I(K,n), and I(Ca) are localized to the basal, synaptic pole of the OHC. Previously we showed that the lateral membrane of the OHC harbors a dense population of voltage sensor-motor elements responsible for OHC motility. This segregation of membrane molecules may have important implications for auditory function. The distribution of OHC conductances will influence the cable properties of the cell, thereby potentially controlling the voltage magnitudes experienced by the motility voltage sensors in the lateral membrane, and thus the output of the "cochlear amplifier."
Acoustic Stimulation↗
Effects of salicylate and lanthanides on outer hair cell motility and associated gating charge.
Salicylate, one of the most widely used drugs, is known to induce reversible tinnitus and hearing loss. Salicylate interferes with outer hair cells (OHCs), which are believed to underlie normal auditory frequency selectivity and sensitivity. In the present experiments, the effects of salicylate and lanthanides on OHC motility and nonlinear capacitance were investigated by using isolated guinea-pig OHCs while attempting to avoid inadvertent intracellular pressure change, which itself can affect OHC motility and capacitance. Either extracellularly or intracellularly applied salicylate reduced nonlinear peak capacitance (Cmpk) and shifted the voltage at peak capacitance to depolarized levels. Concentration-response curves for reduction in Cmpk by salicylate and GdCl3 revealed a half-maximal concentration and Hill coefficient of 1.6 mM and 1.0, and 0.6 mM and 1.2, respectively. In comparable groups of OHCs, the normal Cmpk values of which were near 40 pF, average Cmpk decreased to 28 and 36 pF for intracellularly and extracellularly applied salicylate, respectively. Salicylate reduced, but did not completely block, the voltage-induced length change. Extracellularly, but not intracellularly, applied lanthanide blocked voltage-induced movement and capacitance almost completely. After intracellular trypsin treatment, salicylate reduced voltage-dependent capacitance reversibly, suggesting that salicylate directly acts on the sensor/motor and not via effects on intracellular structures, such as the subsurface cisternae. The results are consistent with the hypothesis that the dissociated, charged form of salicylate directly interacts with the sensor/motor on the inner aspect of the OHC plasma, whereas lanthanides interact on the outer aspect.
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