Electromechanical Models of the Outer Hair Cell Composite Membrane

The outer hair cell (OHC) is an extremely specialized cell and its proper functioning is essential for normal mammalian hearing. This article reviews recent developments in theoretical modeling that have increased our knowledge of the operation of this fascinating cell. The earliest models aimed at capturing experimental observations on voltage-induced cellular length changes and capacitance were based on isotropic elasticity and a two-state Boltzmann function. Recent advances in modeling based on the thermodynamics of orthotropic electroelastic materials better capture the cell’s voltage-dependent stiffness, capacitance, interaction with its environment and ability to generate force at high frequencies. While complete models are crucial, simpler continuum models can be derived that retain fidelity over small changes in transmembrane voltage and strains occurring in vivo. By its function in the cochlea, the OHC behaves like a piezoelectric-like actuator, and the main cellular features can be described by piezoelectric models. However, a finer characterization of the cell’s composite wall requires understanding the local mechanical and electrical fields. One of the key questions is the relative contribution of the in-plane and bending modes of electromechanical strains and forces (moments). The latter mode is associated with the flexoelectric effect in curved membranes. New data, including a novel experiment with tethers pulled from the cell membrane, can help in estimating the role of different modes of electromechanical coupling. Despite considerable progress, many problems still confound modelers. Thus, this article will conclude with a discussion of unanswered questions and highlight directions for future research.     

Power Efficiency of Outer Hair Cell Somatic Electromotility

Cochlear outer hair cells (OHCs) are fast biological motors that serve to enhance the vibration of the organ of Corti and increase the sensitivity of the inner ear to sound. Exactly how OHCs produce useful mechanical power at auditory frequencies, given their intrinsic biophysical properties, has been a subject of considerable debate. To address this we formulated a mathematical model of the OHC based on first principles and analyzed the power conversion efficiency in the frequency domain. The model includes a mixture-composite constitutive model of the active lateral wall and spatially distributed electro-mechanical fields. The analysis predicts that: 1) the peak power efficiency is likely to be tuned to a specific frequency, dependent upon OHC length, and this tuning may contribute to the place principle and frequency selectivity in the cochlea; 2) the OHC power output can be detuned and attenuated by increasing the basal conductance of the cell, a parameter likely controlled by the brain via the efferent system; and 3) power output efficiency is limited by mechanical properties of the load, thus suggesting that impedance of the organ of Corti may be matched regionally to the OHC. The high power efficiency, tuning, and efferent control of outer hair cells are the direct result of biophysical properties of the cells, thus providing the physical basis for the remarkable sensitivity and selectivity of hearing.     

Optimal Electrical Properties of Outer Hair Cells Ensure Cochlear Amplification

The organ of Corti (OC) is the auditory epithelium of the mammalian cochlea comprising sensory hair cells and supporting cells riding on the basilar membrane. The outer hair cells (OHCs) are cellular actuators that amplify small sound-induced vibrations for transmission to the inner hair cells. We developed a finite element model of the OC that incorporates the complex OC geometry and force generation by OHCs originating from active hair bundle motion due to gating of the transducer channels and somatic contractility due to the membrane protein prestin. The model also incorporates realistic OHC electrical properties. It explains the complex vibration modes of the OC and reproduces recent measurements of the phase difference between the top and the bottom surface vibrations of the OC. Simulations of an individual OHC show that the OHC somatic motility lags the hair bundle displacement by ∼90 degrees. Prestin-driven contractions of the OHCs cause the top and bottom surfaces of the OC to move in opposite directions. Combined with the OC mechanics, this results in ∼90 degrees phase difference between the OC top and bottom surface vibration. An appropriate electrical time constant for the OHC membrane is necessary to achieve the phase relationship between OC vibrations and OHC actuations. When the OHC electrical frequency characteristics are too high or too low, the OHCs do not exert force with the correct phase to the OC mechanics so that they cannot amplify. We conclude that the components of OHC forward and reverse transduction are crucial for setting the phase relations needed for amplification.     

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 (V _pkCm ). Rapid phase reversals also occurred at V _pkCm , 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, V _pkCm 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. Received: 31 December 1998/Revised: 12 March 1999     

Fast Electromechanical Amplification in the Lateral Membrane of the Outer Hair Cell

Outer hair cells provide amplification within the mammalian cochlea to enhance audition. The mechanism is believed to reside within the lateral membrane of the cell that houses an expansive array of molecular motors, identified as prestin, which drives somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, at kilohertz rates we have uncovered new details of the early molecular events that arise from voltage perturbations of prestin. We show that dynamic changes in motor state probability occur within the kilohertz range, and signify an amplificatory event. Additionally, we show a lack of effect of Cl driving force, an absence of cell length effect (indicating that the kinetics does not vary across auditory frequency), and the first demonstration of the time dependence of tension induced amplificatory shifts. The process we have identified, where the stimulus-response function shifts in time along the stimulus axis in a multi-exponential manner, bears similarities to those components of adaptation found in the OHC stereociliar transducer identified recently. As with the forward transducer, the speed of the reverse transducer amplificatory event consequently impacts on high frequency peripheral auditory processing.     

Developmental Expression of the Outer Hair Cell Motor Prestin in the Mouse

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C _v), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C _v was about 70% of the mature level. C _v saturated at P18 when hearing shows full maturation. On the other hand, C _lin, which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor’s operating voltage midpoint, V _pkcm, tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V _pkcm reached a steady-state level near −67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V _pkcm, may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.     

Amphipath-Induced Nanoscale Changes in Outer Hair Cell Plasma Membrane Curvature

Outer hair cell (OHC) electromotility enables frequency selectivity and sensitivity in mammalian audition. Electromotility is generated by the transmembrane protein prestin and is sensitive to amphipathic compounds including salicylate, chlorpromazine (CPZ), and trinitrophenol (TNP). Although these compounds induce observable membrane curvature changes in erythrocytes, their effects on OHC membrane curvature are unknown. In this work, fluorescence polarization microscopy was applied to investigate the effects of salicylate, CPZ, and TNP on di-8-ANEPPS orientation in the OHC plasma membrane. Our results demonstrate the ability of fluorescence polarization microscopy to measure amphipath-induced changes in di-8-ANEPPS orientation, consistent with nanoscale changes in membrane curvature between regularly spaced proteins connecting the OHC plasma membrane and cytoskeleton. Simultaneous application of oppositely charged amphipaths generally results in no net membrane bending, consistent with predictions of the bilayer couple hypothesis; however, the application of salicylate (10 mM), which inhibits electromotility, is not reversed by the addition of CPZ. This result supports other findings that suggest salicylate primarily influences electromotiliy and OHC nonlinear capacitance via a direct interaction with prestin. In contrast, we find that CPZ and TNP influence the voltage sensitivity of prestin via membrane bending, demonstrating the mechanosensitivity of this unique membrane motor protein.     

Prestin-Dependence of Outer Hair Cell Survival and Partial Rescue of Outer Hair Cell Loss in Prestin V499G/Y501H Knockin Mice

A knockin (KI) mouse expressing mutated prestin ^V499G/Y501H (499 prestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 prestin OHCs were normal in stiffness and longer than OHCs lacking prestin, accelerated OHC death was unexpectedly observed relative to that documented in prestin knockout (KO) mice. These observations imply an additional role of prestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into prestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 prestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 prestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 prestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 prestin and one copy of KO prestin was also created to reduce quantities of 499 prestin protein. Results show reduction in OHC death in chets, and in 499 prestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 prestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of prestin’s motor function in cochlear amplification.     

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

Compartmentalization of the Outer Hair Cell Demonstrated by Slow Diffusion in the Extracisternal Space

In the outer hair cell (OHC), the extracisternal space (ECiS) is a conduit and reservoir of the molecular and ionic substrates of the lateral wall, including those necessary for electromotility. To determine the mechanisms through which molecules are transported in the ECiS of the OHC, we selectively imaged the time-dependent spatial distribution of fluorescent molecules in a <100 nm layer near the cell/glass interface of the recording chamber after their photolytic activation in a diffraction-limited volume. The effective diffusion coefficient was calculated using the analytical solution of the diffusion equation. It was found that diffusion in the ECiS is isotropic and not affected by depolarizing the OHC. Compared with free solution, the diffusion of 10 kDa dextran was slowed down in both the ECiS and the axial core by a factor of 4.6 and 1.6, respectively.     

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 (Cm _pk ) and shifted the voltage at peak capacitance (V _pkCm ) to hyperpolarized levels. Washout reversed the effects. Perfusion of 0.4 μm TPB⁻ caused an average increase in Cm _pk of 16.3 pF and V _pkCm shift of 13.6 mV. TPP⁺, on the other hand, only shifted V _pkCm in the positive direction, with no change in Cm _pk . 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 (V _{δ Lmax} ) was shifted in correspondence with native V _pkCm , and both changed in a concentration-dependent manner. Both TPB⁻-induced changes in Cm _pk and V _pkCm 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. Received: 14 May 1998/Revised: 24 August 1998     

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.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

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.This revised version was published online in June 2005 with a corrected cover date.     

Optogenetic Control of Mouse Outer Hair Cells

Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects.     

Limiting Frequency of the Cochlear Amplifier Based on Electromotility of Outer Hair Cells

Outer hair cells are the critical element for the sensitivity and sharpness of frequency selectivity of the ear. It is believed that fast motility (electromotility) of these cells is essential for this function. Indeed, force produced by outer hair cells follows their membrane potential very closely at least up to 60 kHz. However, it has been pointed out that the cell's receptor potential is attenuated by a low-pass RC circuit inherent to these cells, with the RC roll-off frequencies significantly lower than their operating frequencies. This would render electromotility ineffective in producing force. To address this issue, we assume that multiple degrees of freedom and vibrational modes due to the complex structure of the organ of Corti provide optimal phases for outer hair cells' force to cancel viscous drag. Our derived frequency limit depends on the drag-capacitance product, not directly on the RC time constant. With a reasonable assumption for the viscous drag, the estimated limit is 10–13 kHz, exceeding the RC corner frequency. Our analysis shows that a fast-activating potassium current can substantially extend the frequency limit by counteracting the capacitive current.     

Hair Cell Interactions in the Statocyst of Hermissenda

Hair cells in the statocyst of Hermissenda crassicornis respond to mechanical stimulation with a short latency (<2 ms) depolarizing generator potential that is followed by hyperpolarization and inhibition of spike activity. Mechanically evoked hyperpolarization and spike inhibition were abolished by cutting the static nerve, repetitive mechanical stimulation, tetrodotoxin (TTX), and Co⁺⁺. Since none of these procedures markedly altered the generator potential it was concluded that the hyperpolarization is an inhibitory synaptic potential and not a component of the mechanotransduction process. Intracellular recordings from pairs of hair cells in the same statocyst and in statocysts on opposite sides of the brain revealed that hair cells are connected by chemical and/or electrical synapses. All chemical interactions were inhibitory. Hyperpolarization and spike inhibition result from inhibitory interactions between hair cells in the same and in opposite statocysts.