Detection of Cochlear Amplification and Its Activation

The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source.     

Detection of Cochlear Amplification and Its Activation

The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source.     

Consequences of Location-Dependent Organ of Corti Micro-Mechanics

The cochlea performs frequency analysis and amplification of sounds. The graded stiffness of the basilar membrane along the cochlear length underlies the frequency-location relationship of the mammalian cochlea. The somatic motility of outer hair cell is central for cochlear amplification. Despite two to three orders of magnitude change in the basilar membrane stiffness, the force capacity of the outer hair cell’s somatic motility, is nearly invariant over the cochlear length. It is puzzling how actuators with a constant force capacity can operate under such a wide stiffness range. We hypothesize that the organ of Corti sets the mechanical conditions so that the outer hair cell’s somatic motility effectively interacts with the media of traveling waves—the basilar membrane and the tectorial membrane. To test this hypothesis, a computational model of the gerbil cochlea was developed that incorporates organ of Corti structural mechanics, cochlear fluid dynamics, and hair cell electro-physiology. The model simulations showed that the micro-mechanical responses of the organ of Corti are different along the cochlear length. For example, the top surface of the organ of Corti vibrated more than the bottom surface at the basal (high frequency) location, but the amplitude ratio was reversed at the apical (low frequency) location. Unlike the basilar membrane stiffness varying by a factor of 1700 along the cochlear length, the stiffness of the organ of Corti complex felt by the outer hair cell remained between 1.5 and 0.4 times the outer hair cell stiffness. The Y-shaped structure in the organ of Corti formed by outer hair cell, Deiters cell and its phalange was the primary determinant of the elastic reactance imposed on the outer hair cells. The stiffness and geometry of the Deiters cell and its phalange affected cochlear amplification differently depending on the location.     

Imaging electrically evoked micromechanical motion within the organ of corti of the excised gerbil cochlea

The outer hair cell (OHC) of the mammalian inner ear exhibits an unusual form of somatic motility that can follow membrane-potential changes at acoustic frequencies. The cellular forces that produce this motility are believed to amplify the motion of the cochlear partition, thereby playing a key role in increasing hearing sensitivity. To better understand the role of OHC somatic motility in cochlear micromechanics, we developed an excised cochlea preparation to visualize simultaneously the electrically-evoked motion of hundreds of cells within the organ of Corti (OC). The motion was captured using stroboscopic video microscopy and quantified using cross-correlation techniques. The OC motion at approximately 2-6 octaves below the characteristic frequency of the region was complex: OHC, Deiter's cell, and Hensen's cell motion were hundreds of times larger than the tectorial membrane, reticular lamina (RL), and pillar cell motion; the inner rows of OHCs moved antiphasic to the outer row; OHCs pivoted about the RL; and Hensen's cells followed the motion of the outer row of OHCs. Our results suggest that the effective stimulus to the inner hair cell hair bundles results not from a simple OC lever action, as assumed by classical models, but by a complex internal motion coupled to the RL.     

The role of organ of Corti mass in passive cochlear tuning

The mechanism for passive cochlear tuning remains unsettled. Early models considered the organ of Corti complex (OCC) as a succession of spring-mass resonators. Later, traveling wave models showed that passive tuning could arise through the interaction of cochlear fluid mass and OCC stiffness without local resonators. However, including enough OCC mass to produce local resonance enhanced the tuning by slowing and thereby growing the traveling wave as it approached its resonant segment. To decide whether the OCC mass plays a role in tuning, the frequency variation of the wavenumber of the cochlear traveling wave was measured (in vivo, passive cochleae) and compared to theoretical predictions. The experimental wavenumber was found by taking the phase difference of basilar membrane motion between two longitudinally spaced locations and dividing by the distance between them. The theoretical wavenumber was a solution of the dispersion relation of a three-dimensional cochlear model with OCC mass and stiffness as the free parameters. The experimental data were only well fit by a model that included OCC mass. However, as the measurement position moved from a best-frequency place of 40 to 12 kHz, the role of mass was diminished. The notion of local resonance seems to only apply in the very high-frequency region of the cochlea.     

Response to a pure tone in a nonlinear mechanical-electrical-acoustical model of the cochlea

In this article, a nonlinear mathematical model is developed based on the physiology of the cochlea of the guinea pig. The three-dimensional intracochlear fluid dynamics are coupled to a micromechanical model of the organ of Corti and to electrical potentials in the cochlear ducts and outer hair cells (OHC). OHC somatic electromotility is modeled by linearized piezoelectric relations whereas the OHC hair-bundle mechanoelectrical transduction current is modeled as a nonlinear function of the hair-bundle deflection. The steady-state response of the cochlea to a single tone is simulated in the frequency domain using an alternating frequency time scheme. Compressive nonlinearity, harmonic distortion, and DC shift on the basilar membrane (BM), tectorial membrane (TM), and OHC potentials are predicted using a single set of parameters. The predictions of the model are verified by comparing simulations to available in vivo experimental data for basal cochlear mechanics. In particular, the model predicts more amplification on the reticular lamina (RL) side of the cochlear partition than on the BM, which replicates recent measurements. Moreover, small harmonic distortion and DC shifts are predicted on the BM, whereas more significant harmonic distortion and DC shifts are predicted in the RL and TM displacements and in the OHC potentials.     

The Spatial Origins of Cochlear Amplification Assessed by Stimulus-Frequency Otoacoustic Emissions

Cochlear amplification of basilar membrane traveling waves is thought to occur between a tone’s characteristic frequency (CF) place and within one octave basal of the CF. Evidence for this view comes only from the cochlear base. Stimulus-frequency otoacoustic emissions (SFOAEs) provide a noninvasive alternative to direct measurements of cochlear motion that can be measured across a wide range of CF regions. Coherent reflection theory indicates that SFOAEs arise mostly from the peak region of the traveling wave, but several studies using far-basal suppressor tones claimed that SFOAE components originate many octaves basal of CF. We measured SFOAEs while perfusing guinea pig cochleas from apex to base with salicylate or KCl solutions that reduced outer-hair-cell function and SFOAE amplification. Solution effects on inner hair cells reduced auditory nerve compound action potentials (CAPs) and provided reference times for when solutions reached the SFOAE-frequency CF region. As solution flowed from apex to base, SFOAE reductions generally occurred later than CAP reductions and showed that the effects of cochlear amplification usually peaked ∼1/2 octave basal of the CF region. For tones ≥2 kHz, cochlear amplification typically extended ∼1.5 octaves basal of CF, and the data are consistent with coherent reflection theory. SFOAE amplification did not extend to the basal end of the cochlea, even though reticular lamina motion is amplified in this region, which indicates that reticular lamina motion is not directly coupled to basilar membrane traveling waves. Previous reports of SFOAE-frequency residuals produced by suppressor frequencies far above the SFOAE frequency are most likely due to additional sources created by the suppressor. For some tones <2 kHz, SFOAE amplification extended two octaves apical of CF, which highlights that different vibratory motions produce SFOAEs and CAPs, and that the amplification region depends on the cochlear mode of motion considered. The concept that there is a single “cochlear amplification region” needs to be revised.     

Coupling active hair bundle mechanics, fast adaptation, and somatic motility in a cochlear model

One of the central questions in the biophysics of the mammalian cochlea is determining the contributions of the two active processes, prestin-based somatic motility and hair bundle (HB) motility, to cochlear amplification. HB force generation is linked to fast adaptation of the transduction current via a calcium-dependent process and somatic force generation is driven by the depolarization caused by the transduction current. In this article, we construct a global mechanical-electrical-acoustical mathematical model of the cochlea based on a three-dimensional fluid representation. The global cochlear model is coupled to linearizations of nonlinear somatic motility and HB activity as well as to the micromechanics of the passive structural and electrical elements of the cochlea. We find that the active HB force alone is not sufficient to power high frequency cochlear amplification. However, somatic motility can overcome resistor-capacitor filtering by the basolateral membrane and deliver sufficient mechanical energy for amplification at basal locations. The results suggest a new theory for high frequency active cochlear mechanics, in which fast adaptation controls the transduction channel sensitivity and thereby the magnitude of the energy delivered by somatic motility.     

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.     

Model of cochlear microphonic explores the tuning and magnitude of hair cell transduction current

The mammalian cochlea relies on the active forcing of sensory outer hair cells (OHCs) to amplify traveling wave responses along the basilar membrane. These forces are the result of electromotility, wherein current through the OHCs leads to conformational changes in the cells that provide stresses on surrounding structures. OHC transducer current can be detected via the voltage in the scala tympani (the cochlear microphonic, CM), and the CM can be used as an indicator of healthy cochlear operation. The CM represents a summation of OHC currents (the inner hair cell contribution is known to be small) and to use CM to probe the properties of OHC transduction requires a model that simulates that summation. We developed a finite element model for that purpose. The pattern of current generators (the model input) was initially based on basilar membrane displacement, with the current size based on in vitro data. The model was able to reproduce the amplitude of experimental CM results reasonably well when the input tuning was enhanced slightly (peak increased by ∼6 dB), which can be regarded as additional hair bundle tuning, and with a current/input value of 200–260 pA/nm, which is ∼4 times greater than the largest in vitro measures.     

Outer hair cell somatic electromotility in vivo and power transfer to the organ of Corti

The active amplification of sound-induced vibrations in the cochlea, known to be crucial for auditory sensitivity and frequency selectivity, is not well understood. The outer hair cell (OHC) somatic electromotility is a potential mechanism for such amplification. Its effectiveness in vivo is putatively limited by the electrical low-pass filtering of the cell's transmembrane potential. However, the transmembrane potential is an incomplete metric. We propose and estimate two metrics to evaluate the effectiveness of OHC electromotility in vivo. One metric is the OHC electromechanical ratio defined as the amplitude of the ratio of OHC displacement to the change in its transmembrane potential. The in vivo electromechanical ratio is derived from the recently measured in vivo displacements of the reticular lamina and the basilar membrane at the 19 kHz characteristic place in guinea pigs and using a model. The ratio, after accounting for the differences in OHC vibration in situ due to the impedances from the adjacent structures, is in agreement with the literature values of the in vitro electromechanical ratio measured by others. The second and more insightful metric is the OHC somatic power. Our analysis demonstrates that the organ of Corti is nearly optimized to receive maximum somatic power in vivo and that the estimated somatic power could account for the active amplification.     

Chloride-driven Electromechanical Phase Lags at Acoustic Frequencies Are Generated by SLC26a5, the Outer Hair Cell Motor Protein

Outer hair cells (OHC) possess voltage-dependent membrane bound molecular motors, identified as the solute carrier protein SLC26a5, that drive somatic motility at acoustic frequencies. The electromotility (eM) of OHCs provides for cochlear amplification, a process that enhances auditory sensitivity by up to three orders of magnitude. In this study, using whole cell voltage clamp and mechanical measurement techniques, we identify disparities between voltage sensing and eM that result from stretched exponential electromechanical behavior of SLC26a5, also known as prestin, for its fast responsiveness. This stretched exponential behavior, which we accurately recapitulate with a new kinetic model, the meno presto model of prestin, influences the protein’s responsiveness to chloride binding and provides for delays in eM relative to membrane voltage driving force. The model predicts that in the frequency domain, these delays would result in eM phase lags that we confirm by measuring OHC eM at acoustic frequencies. These lags may contribute to canceling viscous drag, a requirement for many models of cochlear amplification.     

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".     

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.     

Power Dissipation in the Cochlea Can Enhance Frequency Selectivity

The cochlear cavity is filled with viscous fluids, and it is partitioned by a viscoelastic structure called the organ of Corti complex. Acoustic energy propagates toward the apex of the cochlea through vibrations of the organ of Corti complex. The dimensions of the vibrating structures range from a few hundred (e.g., the basilar membrane) to a few micrometers (e.g., the stereocilia bundle). Vibrations of microstructures in viscous fluid are subjected to energy dissipation. Because the viscous dissipation is considered to be detrimental to the function of hearing—sound amplification and frequency tuning—the cochlea uses cellular actuators to overcome the dissipation. Compared to extensive investigations on the cellular actuators, the dissipating mechanisms have not been given appropriate attention, and there is little consensus on damping models. For example, many theoretical studies use an inviscid fluid approximation and lump the viscous effect to viscous damping components. Others neglect viscous dissipation in the organ of Corti but consider fluid viscosity. We have developed a computational model of the cochlea that incorporates viscous fluid dynamics, organ of Corti microstructural mechanics, and electrophysiology of the outer hair cells. The model is validated by comparing with existing measurements, such as the viscoelastic response of the tectorial membrane, and the cochlear input impedance. Using the model, we investigated how dissipation components in the cochlea affect its function. We found that the majority of acoustic energy dissipation of the cochlea occurs within the organ of Corti complex, not in the scalar fluids. Our model suggests that an appropriate dissipation can enhance the tuning quality by reducing the spread of energy provided by the outer hair cells’ somatic motility.     

Pivotal role of actin depolymerization in the regulation of cochlear outer hair cell motility

Cochlear outer hair cells undergo reversible changes in shape when externally stimulated. This response, known as OHC motility, is a central component of the cochlear amplifier, the mechanism responsible for the high sensitivity of mammalian hearing. We report that actin depolymerization, as regulated by activation/inhibition of LIMK/cofilin-mediated pathways, has a pivotal role in OHC motility. LIMK-mediated cofilin phosphorylation, which inhibits the actin depolymerizing activity of this protein, increases both electromotile amplitude and total length of guinea pig OHCs. In contrast, a decrease in cofilin phosphorylation reduces both OHC electromotile amplitude and OHC length. Experiments with acetylcholine and lysophosphatidic acid indicate that the effects of these agents on OHC motility are associated with regulation of cofilin phosphorylation via different signaling cascades. On the other hand, nonlinear capacitance measurements confirmed that all observed changes in OHC motile response were independent of the performance of the motor protein prestin. Altogether, these results strongly support the hypothesis that the cytoskeleton has a major role in the regulation of OHC motility, and identify actin depolymerization as a key process for modulating cochlear amplification.     

Experimental and Theoretical Explorations of Traveling Waves and Tuning in the Bushcricket Ear

The ability to detect airborne sound is essential for many animals. Examples from the inner ear of mammals and bushcrickets demonstrate that similar detection strategies evolved in taxonomically distant species. Both mammalian and bushcricket ears possess a narrow strip of sensory tissue that exhibits an anatomical gradient and traveling wave motion responses used for frequency discrimination. We measured pressure and motion in the bushcricket ear to investigate physical properties, stiffness, and mass, which govern the mechanical responses to sound. As in the mammalian cochlea, sound-induced fluid pressure and motion responses were tonotopically organized along the longitudinal axis of the crista acustica, the bushcricket’s hearing organ. The fluid pressure at the crista and crista motion were used to calculate the acoustic impedance of the organ-bounded fluid mass (Z_mass). We used a theoretical wave analysis of wavelength data from a previous study to predict the crista acustica stiffness. The wave analysis also predicts Z_mass, and that result agreed reasonably well with the directly measured Z_mass, lending support to the theoretical wave analysis. The magnitude of the crista stiffness was similar to basilar membrane stiffness in mammals, and as in mammals, the stiffness decreased from the high-frequency to the low-frequency region. At a given location, the stiffness increased with increasing frequency, corresponding to increasing curvature of the traveling wave (decreasing wavelength), indicating that longitudinal coupling plays a substantial role in determining crista stiffness. This is in contrast to the mammalian ear, in which stiffness is independent of frequency and longitudinal coupling is relatively small.     

Genome-wide analysis of chicken snoRNAs provides unique implications for the evolution of vertebrate snoRNAs

Background

Although outer hair cells (OHCs) play a key role in cochlear amplification, it is not fully understood how they amplify sound signals by more than 100 fold. Two competing or possibly complementary mechanisms, stereocilia-based and somatic electromotility-based amplification, have been considered. Lacking knowledge about the exceptionally rich protein networks in the OHC plasma membrane, as well as related protein-protein interactions, limits our understanding of cochlear function. Therefore, we focused on finding protein partners for two important membrane proteins: Cadherin 23 (cdh23) and prestin. Cdh23 is one of the tip-link proteins involved in transducer function, a key component of mechanoelectrical transduction and stereocilia-based amplification. Prestin is a basolateral membrane protein responsible for OHC somatic electromotility.

Results

Using the membrane-based yeast two-hybrid system to screen a newly built cDNA library made predominantly from OHCs, we identified two completely different groups of potential protein partners using prestin and cdh23 as bait. These include both membrane bound and cytoplasmic proteins with 12 being de novo gene products with unknown function(s). In addition, some of these genes are closely associated with deafness loci, implying a potentially important role in hearing. The most abundant prey for prestin (38%) is composed of a group of proteins involved in electron transport, which may play a role in OHC survival. The most abundant group of cdh23 prey (55%) contains calcium-binding domains. Since calcium performs an important role in hair cell mechanoelectrical transduction and amplification, understanding the interactions between cdh23 and calcium-binding proteins should increase our knowledge of hair cell function at the molecular level.

Conclusion

The results of this study shed light on some protein networks in cochlear hair cells. Not only was a group of de novo genes closely associated with known deafness loci identified, but the data also indicate that the hair cell tip link interacts directly with calcium binding proteins. The OHC motor protein, prestin, also appears to be associated with electron transport proteins. These unanticipated results open potentially fruitful lines of investigation into the molecular basis of cochlear amplification.     

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin–CASK interaction at the basal junction with the Deiter’s cell

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter’s cell may contribute to efficient generation of somatic electromechanical force.     

Chloride-driven Electromechanical Phase Lags at Acoustic Frequencies Are Generated by SLC26a5, the Outer Hair Cell Motor Protein

Outer hair cells (OHC) possess voltage-dependent membrane bound molecular motors, identified as the solute carrier protein SLC26a5, that drive somatic motility at acoustic frequencies. The electromotility (eM) of OHCs provides for cochlear amplification, a process that enhances auditory sensitivity by up to three orders of magnitude. In this study, using whole cell voltage clamp and mechanical measurement techniques, we identify disparities between voltage sensing and eM that result from stretched exponential electromechanical behavior of SLC26a5, also known as prestin, for its fast responsiveness. This stretched exponential behavior, which we accurately recapitulate with a new kinetic model, the meno presto model of prestin, influences the protein’s responsiveness to chloride binding and provides for delays in eM relative to membrane voltage driving force. The model predicts that in the frequency domain, these delays would result in eM phase lags that we confirm by measuring OHC eM at acoustic frequencies. These lags may contribute to canceling viscous drag, a requirement for many models of cochlear amplification.The outer hair cell (OHC) is one of two receptor cell types in the organ of Corti, but unlike the inner hair cell it displays electromotile behavior distinct from any other form of cellular motility (1–4). OHC electromotility (eM) arises from the concerted action of millions of molecular motors embedded in the lateral membrane of the cell. They respond directly to membrane voltage and evidence reciprocal activity; namely, they are piezoelectric-like (5–7). Indeed, there is clear evidence that surface area changes accompany state transitions in the motor [see (8)]. The identification of these motors as members of the anion transporter family SLC26 (9), of which prestin is the 5th member (a5), underscores an interesting molecular evolution designed to boost the performance of auditory sensitivity and selectivity. This enhancement is known as cochlear amplification (10).A class of cochlear models requires an electromechanical phase disparity for effective cochlear amplification (11–13), OHC eM lagging receptor potentials. Traditionally, these models assign the mechanism to processes other than the OHC itself. The phase lag provides for the properly timed injection of mechanical force into the cochlear partition to counter viscous detriment. Most molecular models of prestin behavior envision tightly coupled interactions between membrane voltage and eM, arising from sensor charge movements obeying Boltzmann statistics (14–20). Thus, Boltzmann characteristics of sensor charge and eM, namely Q_max /eM_max and Q V_h / eM V_h, are commonly believed to tightly correspond. However, we recently showed significant uncoupling of these characteristics depending on rate and polarity of voltage stimulation and on intracellular chloride level (21). We showed that a slow intermediate transition placed between prestin’s chloride binding transition and the voltage dependent transition responsible for eM could qualitatively account for the data, and we surmised that a molecularly based phase lag should arise. In this study we test this hypothesis by measuring eM at acoustic frequencies and find that indeed substantial frequency dependent phase lags are produced between membrane voltage and eM, showing chloride dependence. An enhanced stretched-exponential kinetic model, termed the meno presto model of prestin, nicely fits the data, whereas a model lacking the intermediate transitions fails.