Outer Hair Cell Electromotility Research Articles

Characterization of Hair Cell-Like Cells Converted From Supporting Cells After Notch Inhibition in Cultures of the Organ of Corti From Neonatal Gerbils.

The senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Hair cells transduce mechanical stimuli into electrical activity. Loss of hair cells as a result of aging or exposure to noise and ototoxic drugs is the major cause of noncongenital hearing and balance deficits. In the ear of non-mammals, lost hair cells can spontaneously be replaced by production of new hair cells from conversion of supporting cells. Although supporting cells in adult mammals have lost that capability, neonatal supporting cells are able to convert to hair cells after inhibition of Notch signaling. We questioned whether Notch inhibition is sufficient to convert supporting cells to functional hair cells using electrophysiology and electron microscopy. We showed that pharmacological inhibition of the canonical Notch pathway in the cultured organ of Corti prepared from neonatal gerbils induced stereocilia formation in supporting cells (defined as hair cell-like cells or HCLCs) and supernumerary stereocilia in hair cells. The newly emerged stereocilia bundles of HCLCs were functional, i.e., able to respond to mechanical stimulation with mechanotransduction (MET) current. Transmission electron microscopy (TEM) showed that HCLCs converted from pillar cells maintained the pillar cell shape and that subsurface cisternae, normally observed underneath the cytoskeleton in outer hair cells (OHCs), was not present in Deiters’ cells-derived HCLCs. Voltage-clamp recordings showed that whole-cell currents from Deiters’ cells-derived HCLCs retained the same kinetics and magnitude seen in normal Deiters’ cells and that nonlinear capacitance (NLC), an electrical hallmark of OHC electromotility, was not detected from any HCLCs measured. Taken together, these results suggest that while Notch inhibition is sufficient for promoting stereocilia bundle formation, it is insufficient to convert neonatal supporting cells to mature hair cells. The fact that Notch inhibition led to stereocilia formation in supporting cells and supernumerary stereocilia in existing hair cells appears to suggest that Notch signaling may regulate stereocilia formation and stability during development.

Efferent modulation of pre-neural and neural distortion products

Distortion product otoacoustic emissions (DPOAEs) and distortion product frequency following responses (DPFFRs) are respectively pre-neural and neural measurements associated with cochlear nonlinearity. Because cochlear nonlinearity is putatively linked to outer hair cell electromotility, DPOAEs and DPFFRs may provide complementary measurements of the human medial olivocochlear (MOC) reflex, which directly modulates outer hair cell function. In this study, we first quantified MOC reflex-induced DPOAE inhibition at spectral fine structure peaks in 22 young human adults with normal hearing. The f1 and f2 tone pairs producing the largest DPOAE fine structure peak for each subject were then used to evoke DPFFRs with and without MOC reflex activation to provide a related neural measure of efferent inhibition. We observed significant positive relationships between DPOAE fine structure peak inhibition and inhibition of DPFFR components representing neural phase locking to f2 and 2f1-f2, but not f1. These findings may support previous observations that the MOC reflex inhibits DPOAE sources differentially. That these effects are maintained and represented in the auditory brainstem suggests that the MOC reflex may exert a potent influence on subsequent subcortical neural representation of sound.

Diflunisal inhibits prestin by chloride-dependent mechanism.

The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.

Spontaneous otoacoustic emission recordings during contralateral pure-tone activation of medial olivocochlear reflex.

We hypothesized that cochlear frequency discrimination occurs through medial olivocochlear efferent (MOCE)-induced alterations in outer hair cell (OHC) electromotility, which is independent from basilar membrane traveling waves. After obtaining informed consent, volunteers with normal hearing (n = 10; mean age: 20.6±1.2 years) and patients with unilateral deafness (n = 10; mean age: 30.2±17.9 years) or bilateral deafness (n = 8; mean age: 30.7±13.8 years) underwent a complete physical and audiological examination, and audiological tests including transient evoked otoacoustic emission and spontaneous otoacoustic emission (TEOAE and SOAE, respectively). SOAE recordings were performed during contralateral pure-tone stimuli at 1 and 3kHz. SOAE recordings in the presence of contralateral pure-tone stimuli showed frequency-specific activation out of the initial frequency range of SOAE responses. Basilar membrane motion during pure-tone stimulation results from OHC activation by means of MOCE neurons rather than from a traveling wave. Eventually, frequency-specific responses obtained from SOAEs suggested that OHC electromotility may be responsible for frequency discrimination of the cochlea independently from basilar membrane motion.

Progressive age-dependence and frequency difference in the effect of gap junctions on active cochlear amplification and hearing

Mutations of Connexin 26 (Cx26, GJB2), which is a predominant gap junction isoform in the cochlea, can induce high incidence of nonsyndromic hearing loss. We previously found that targeted-deletion of Cx26 in supporting Deiters cells and outer pillar cells in the cochlea can influence outer hair cell (OHC) electromotility and reduce active cochlear amplification leading to hearing loss, even though there are no gap junction connexin expressions in the auditory sensory hair cells. Here, we further report that hearing loss and the reduction of active amplification in the Cx26 targeted-deletion mice are progressive and different at high and low frequency regions, first occurring in the high frequency region and then progressively extending to the middle and low frequency regions with mouse age increased. The speed of hearing loss extending was fast in the basal high frequency region and slow in the apical low frequency region, showing a logarithmic function with mouse age. Before postnatal day 25, there were no significant hearing loss and the reduction of active cochlear amplification in the low frequency region. Hearing loss and the reduction of active cochlear amplification also had frequency difference, severe and large in the high frequency regions. These new data indicate that the effect of gap junction on active cochlear amplification is progressive, but, consistent with our previous report, exists in both high and low frequency regions in adulthood. These new data also suggest that cochlear gap junctions may have an important role in age-related hearing loss.

A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics

Accelerated age-related hearing loss disrupts high-frequency hearing in inbred CD-1 mice. The p.Ala88Val (A88V) mutation in the gene coding for the gap-junction protein connexin30 (Cx30) protects the cochlear basal turn of adult CD-1Cx30A88V/A88V mice from degeneration and rescues hearing. Here we report that the passive compliance of the cochlear partition and active frequency tuning of the basilar membrane are enhanced in the cochleae of CD-1Cx30A88V/A88V compared to CBA/J mice with sensitive high-frequency hearing, suggesting that gap junctions contribute to passive cochlear mechanics and energy distribution in the active cochlea. Surprisingly, the endocochlear potential that drives mechanoelectrical transduction currents in outer hair cells and hence cochlear amplification is greatly reduced in CD-1Cx30A88V/A88V mice. Yet, the saturating amplitudes of cochlear microphonic potentials in CD-1Cx30A88V/A88V and CBA/J mice are comparable. Although not conclusive, these results are compatible with the proposal that transmembrane potentials, determined mainly by extracellular potentials, drive somatic electromotility of outer hair cells.

Ablation of the auditory cortex results in changes in the expression of neurotransmission-related mRNAs in the cochlea

The auditory cortex (AC) dynamically regulates responses of the Organ of Corti to sound through descending connections to both the medial (MOC) and lateral (LOC) olivocochlear efferent systems. We have recently provided evidence that AC has a reinforcement role in the responses to sound of the auditory brainstem nuclei. In a molecular level, we have shown that descending inputs from AC are needed to regulate the expression of molecules involved in outer hair cell (OHC) electromotility control, such as prestin and the α10 nicotinic acetylcholine receptor (nAchR). In this report, we show that descending connections from AC to olivocochlear neurons are necessary to regulate the expression of molecules involved in cochlear afferent signaling. RT-qPCR was performed in rats at 1, 7 and 15 days after unilateral ablation of the AC, and analyzed the time course changes in gene transcripts involved in neurotransmission at the first auditory synapse. This included the glutamate metabolism enzyme glutamate decarboxylase 1 (glud1) and AMPA glutamate receptor subunits GluA2-4. In addition, gene transcripts involved in efferent regulation of type I spiral ganglion neuron (SGN) excitability mediated by LOC, such as the α7 nAchR, the D2 dopamine receptor, and the α1, and γ2 GABAA receptor subunits, were also investigated. Unilateral AC ablation induced up-regulation of GluA3 receptor subunit transcripts, whereas both GluA2 and GluA4 mRNA receptors were down-regulated already at 1 day after the ablation. Unilateral removal of the AC also resulted in up-regulation of the transcripts for α7 nAchR subunit, D2 dopamine receptor, and α1 GABAA receptor subunit at 1 day after the ablation. Fifteen days after the injury, AC ablations induced an up-regulation of glud1 transcripts.

Energy Output from a Single Outer Hair Cell

Electromotility of outer hair cells (OHCs) has been extensively studied with in vitro experiments because of its physiological significance to the cochlear amplifier, which provides the exquisite sensitivity and frequency selectivity of the mammalian ear. However, these studies have been performed largely under load-free conditions or with static load, while these cells function in vivo in a dynamic environment, receiving electrical energy to enhance mechanical oscillation in the inner ear. This gap leaves uncertainties in addressing a key issue, how much mechanical energy an OHC provides. This study is an attempt of bridging the gap by introducing a simple one-dimensional model for electromotility of OHC in a dynamic environment. This model incorporates a feedback loop involving the receptor potential and the mechanical load on OHC, and leads to an analytical expression for the membrane capacitance, which explicitly describes the dependence on the elastic load, viscous drag, and the mass. The derived equation of motion was examined in a mass-less model system with realistic parameter values for OHC. It was found that viscous drag is more effective than elastic load in enhancing the receptor potential that drives the cell. For this reason, it is expected that OHCs are more effective in counteracting viscous drag than providing elastic energy to the system.

Editorial: Auditory Efferent System: New Insights from Cortex to Cochlea.

Editorial: Auditory Efferent System: New Insights from Cortex to Cochlea.

Chloride Anions Regulate Kinetics but Not Voltage-Sensor Qmax of the Solute Carrier SLC26a5

In general, SLC26 solute carriers serve to transport a variety of anions across biological membranes. However, prestin (SLC26a5) has evolved, now serving as a motor protein in outer hair cells (OHCs) of the mammalian inner ear and is required for cochlear amplification, a mechanical feedback mechanism to boost auditory performance. The mechanical activity of the OHC imparted by prestin is driven by voltage and controlled by anions, chiefly intracellular chloride. Current opinion is that chloride anions control the Boltzmann characteristics of the voltage sensor responsible for prestin activity, including Qmax, the total sensor charge moved within the membrane, and Vh, a measure of prestin’s operating voltage range. Here, we show that standard narrow-band, high-frequency admittance measures of nonlinear capacitance (NLC), an alternate representation of the sensor’s charge-voltage (Q-V) relationship, is inadequate for assessment of Qmax, an estimate of the sum of unitary charges contributed by all voltage sensors within the membrane. Prestin’s slow transition rates and chloride-binding kinetics adversely influence these estimates, contributing to the prevalent concept that intracellular chloride level controls the quantity of sensor charge moved. By monitoring charge movement across frequency, using measures of multifrequency admittance, expanded displacement current integration, and OHC electromotility, we find that chloride influences prestin kinetics, thereby controlling charge magnitude at any particular frequency of interrogation. Importantly, however, this chloride dependence vanishes as frequency decreases, with Qmax asymptoting at a level irrespective of the chloride level. These data indicate that prestin activity is significantly low-pass in the frequency domain, with important implications for cochlear amplification. We also note that the occurrence of voltage-dependent charge movements in other SLC26 family members may be hidden by inadequate interrogation timescales, and that revelation of such activity could highlight an evolutionary means for kinetic modifications within the family to address hearing requirements in mammals.

A Walkthrough of Nonlinear Capacitance Measurement of Outer Hair Cells.

Nonlinear capacitance (NLC) measures are often used as surrogate measures of outer hair cell (OHC) electromotility (eM), since the two are commonly thought to share many biophysical features. The measurement of NLC is simpler than direct measurements of eM and, therefore, many investigators have adopted it. A standard patch-clamp hardware configuration is sufficient for recording NLC, given the proper software interface. Thus, the approach is cost effective. We use the software jClamp since it is tailored to capacitance measurement. Here we detail steps that we use to measure NLC. The walk through includes isolation of guinea pig OHCs, building voltage commands, recording, and analysis.

Synchronized Progression of Prestin Expression and Auditory Brainstem Response during Postnatal Development in Rats.

Prestin is the motor protein expressed in the cochlear outer hair cells (OHCs) of mammalian inner ear. The electromotility of OHCs driven by prestin is responsible for the cochlear amplification which is required for normal hearing in adult animals. Postnatal expression of prestin and activity of OHCs may contribute to the maturation of hearing in rodents. However, the temporal and spatial expression of prestin in cochlea during the development is not well characterized. In the present study, we examined the expression and function of prestin from the OHCs in apical, middle, and basal turns of the cochleae of postnatal rats. Prestin first appeared at postnatal day 6 (P6) for basal turn, P7 in middle turn, and P9 for apical turn of cochlea. The expression level increased progressively over the next few days and by P14 reached the mature level for all three segments. By comparison with the time course of the development of auditory brainstem response for different frequencies, our data reveal that prestin expression synchronized with the hearing development. The present study suggests that the onset time of hearing may require the expression of prestin and is determined by the mature function of OHCs.

Auditory Pathology in a Transgenic mtTFB1 Mouse Model of Mitochondrial Deafness

The A1555G mutation in the 12S rRNA gene of human mitochondrial DNA causes maternally inherited, nonsyndromic deafness, an extreme case of tissue-specific mitochondrial pathology. A transgenic mouse strain that robustly overexpresses the mitochondrial 12S ribosomal RNA methyltransferase TFB1M (Tg-mtTFB1 mice) exhibits progressive hearing loss that we proposed models aspects of A1555G-related pathology in humans. Although our previous studies of Tg-mtTFB1 mice implicated apoptosis in the spiral ganglion and stria vascularis because of mitochondrial reactive oxygen species-mediated activation of AMP kinase (AMPK) and the nuclear transcription factor E2F1, detailed auditory pathology was not delineated. Herein, we show that Tg-mtTFB1 mice have reduced endocochlear potential, indicative of significant stria vascularis dysfunction, but without obvious signs of strial atrophy. We also observed decreased auditory brainstem response peak 1 amplitude and prolonged wave I latency, consistent with apoptosis of spiral ganglion neurons. Although no major loss of hair cells was observed, there was a mild impairment of voltage-dependent electromotility of outer hair cells. On the basis of these results, we propose that these events conspire to produce the progressive hearing loss phenotype in Tg-mtTFB1 mice. Finally, genetically reducing AMPK α1 rescues hearing loss in Tg-mtTFB1 mice, confirming that aberrant up-regulation of AMPK signaling promotes the observed auditory pathology. The relevance of these findings to human A1555G patients and the potential therapeutic value of reducing AMPK activity are discussed.

Activity-dependent regulation of prestin expression in mouse outer hair cells.

Prestin is a membrane protein necessary for outer hair cell (OHC) electromotility and normal hearing. Its regulatory mechanisms are unknown. Several mouse models of hearing loss demonstrate increased prestin, inspiring us to investigate how hearing loss might feedback onto OHCs. To test whether centrally mediated feedback regulates prestin, we developed a novel model of inner hair cell loss. Injection of diphtheria toxin (DT) into adult CBA mice produced significant loss of inner hair cells without affecting OHCs. Thus, DT-injected mice were deaf because they had no afferent auditory input despite OHCs continuing to receive normal auditory mechanical stimulation and having normal function. Patch-clamp experiments demonstrated no change in OHC prestin, indicating that loss of information transfer centrally did not alter prestin expression. To test whether local mechanical feedback regulates prestin, we used Tecta(C1509G) mice, where the tectorial membrane is malformed and only some OHCs are stimulated. OHCs connected to the tectorial membrane had normal prestin levels, whereas OHCs not connected to the tectorial membrane had elevated prestin levels, supporting an activity-dependent model. To test whether the endocochlear potential was necessary for prestin regulation, we studied Tecta(C1509G) mice at different developmental ages. OHCs not connected to the tectorial membrane had lower than normal prestin levels before the onset of the endocochlear potential and higher than normal prestin levels after the onset of the endocochlear potential. Taken together, these data indicate that OHC prestin levels are regulated through local feedback that requires mechanoelectrical transduction currents. This adaptation may serve to compensate for variations in the local mechanical environment.

Acoustic input and efferent activity regulate the expression of molecules involved in cochlear micromechanics.

Electromotile activity in auditory outer hair cells (OHCs) is essential for sound amplification. It relies on the highly specialized membrane motor protein prestin, and its interactions with the cytoskeleton. It is believed that the expression of prestin and related molecules involved in OHC electromotility may be dynamically regulated by signals from the acoustic environment. However little is known about the nature of such signals and how they affect the expression of molecules involved in electromotility in OHCs. We show evidence that prestin oligomerization is regulated, both at short and relatively long term, by acoustic input and descending efferent activity originating in the cortex, likely acting in concert. Unilateral removal of the middle ear ossicular chain reduces levels of trimeric prestin, particularly in the cochlea from the side of the lesion, whereas monomeric and dimeric forms are maintained or even increased in particular in the contralateral side, as shown in Western blots. Unilateral removal of the auditory cortex (AC), which likely causes an imbalance in descending efferent activity on the cochlea, also reduces levels of trimeric and tetrameric forms of prestin in the side ipsilateral to the lesion, whereas in the contralateral side prestin remains unaffected, or even increased in the case of trimeric and tetrameric forms. As far as efferent inputs are concerned, unilateral ablation of the AC up-regulates the expression of α10 nicotinic Ach receptor (nAChR) transcripts in the cochlea, as shown by RT-Quantitative real-time PCR (qPCR). This suggests that homeostatic synaptic scaling mechanisms may be involved in dynamically regulating OHC electromotility by medial olivocochlear efferents. Limited, unbalanced efferent activity after unilateral AC removal, also affects prestin and β-actin mRNA levels. These findings support that the concerted action of acoustic and efferent inputs to the cochlea is needed to regulate the expression of major molecules involved in OHC electromotility, both at the transcriptional and posttranscriptional levels.

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.

Localization of kainate receptors in inner and outer hair cell synapses

Glutamate plays a role in hair cell afferent transmission, but the receptors that mediate neurotransmission between outer hair cells (OHCs) and type II ganglion neurons are not well defined. A previous study using in situ hybridization showed that several kainate-type glutamate receptor (KAR) subunits are expressed in cochlear ganglion neurons. To determine whether KARs are expressed in hair cell synapses, we performed X-gal staining on mice expressing lacZ driven by the GluK5 promoter, and immunolabeling of glutamate receptors in whole-mount mammalian cochleae. X-gal staining revealed GluK5 expression in both type I and type II ganglion neurons and OHCs in adults. OHCs showed X-gal reactivity throughout maturation from postnatal day 4 (P4) to 1.5 months. Immunoreactivity for GluK5 in IHC afferent synapses appeared to be postsynaptic, similar to GluA2 (GluR2; AMPA-type glutamate receptor (AMPAR) subunit), while GluK2 may be on both sides of the synapses. In OHC afferent synapses, immunoreactivity for GluK2 and GluK5 was found, although GluK2 was only in those synapses bearing ribbons. GluA2 was not detected in adult OHC afferent synapses. Interestingly, GluK1, GluK2 and GluK5 were also detected in OHC efferent synapses, forming several active zones in each synaptic area. At P8, GluA2 and all KAR subunits except GluK4 were detected in OHC afferent synapses in the apical turn, and GluA2, GluK1, GluK3 decreased dramatically in the basal turn. These results indicate that AMPARs and KARs (GluK2/GluK5) are localized to IHC afferent synapses, while only KARs (GluK2/GluK5) are localized to OHC afferent synapses in adults. Glutamate spillover near OHCs may act on KARs in OHC efferent terminals to modulate transmission of acoustic information and OHC electromotility.

Effect of General Anesthetics and Alcohols on Prestin (SLC26A5) Function

Prestin is a membrane protein essential to the electromotility of outer-hair cells (OHC) in the cochlea. This protein with piezoelectric properties has shown sensitivity to changes in cell membrane physical properties. The presence of molecules which impact the lateral pressure or the fluidity of the membrane -such as cholesterol, NSAIDs or lipophilic ions- can alter the electrophysiological properties of prestin as well as the electromotility. In an OHC, the nonlinear capacitance (NLC) and the electromotility are both conferred by prestin and coupled. This allows monitoring the NLC as a surrogate measure of prestin's function.Here we describe the impact of general anesthetics and alcohols on prestin, and aim to use this protein as a model system for studying membrane-protein interaction through changes in the NLC. We tested the effect on prestin of alcohols with various C-chains (C2 to C10) and some general anesthetics (GA: propofol, isoflurane, halothane, chloroform, etomidate and xylazine). All the alcohols tested trigger a dose-dependent shift in the characteristic voltage at half-maximal charge transfer (V1/2), with the sensitivity increasing as the alcohols' carbon-chain is longer. The direction of the shift changes with concentration, the lower concentration cause a negative shift while higher concentrations shift V1/2 closer to 0mV. These shifts are correlated with changes in the linear capacitance of the cell. All of the GA we tested caused a dose-dependent increase in charge density at sub-millimolar concentrations as well as a shift of V1/2 toward hyperpolarized voltages. The sensitivity of prestin to these molecules seems to follow the Meyer/Overton correlation.The shift in the NLC as well as the increase in charge density can modify the function of the OHC at resting potential and alter cochlear amplification.

Active cochlear amplification is dependent on supporting cell gap junctions

Mammalian hearing relies upon active cochlear mechanics, which arises from outer hair cell (OHC) electromotility and hair bundle movement, to amplify acoustic stimulations increasing hearing sensitivity and frequency selectivity. Here we describe the novel finding that gap junctions between cochlear supporting cells also have a critical role in active cochlear amplification in vivo. We find that targeted-deletion of connexin26 (Cx26) in Deiters cells (DCs) and outer pillar cells (OPCs), which constrain OHCs standing on the basilar membrane, causes a leftward shift in OHC electromotility towards hyperpolarization, and reduces active cochlear amplification with hearing loss. Coincident with large reduction in distortion product otoacoustic emission (DPOAE) and severe hearing loss at high frequencies, the shift is larger in shorter OHCs. Our study demonstrates that active cochlear amplification in vivo is dependent on supporting cell gap junctions. These new findings also show that Cx26 deficiency can reduce active cochlear amplification to induce hearing loss.

Effect of Non-Steroidal Anti-Inflamatory Drugs on the Outer-Hair Cell Protein Prestin

Prestin belongs to the SLC26 protein family of anion transporters. Prestin (SLC26A5) is a motor protein essential for the electromotility of the outer hair- cells (OHC) and therefore the amplification of sound in the cochlea. This protein is able to convert changes in membrane potential to mechanical force and to modify the length of the OHC. The electromotility of prestin-expressing cells is associated with a nonlinear capacitance (NLC) that can be measured electrophysiologically. Prestin is a mechanosensitive protein and its function is altered by reagents known to change membrane mechanical properties.The non-steroidal anti-inflammatory drug (NSAID) salicylate has been showed to inhibit the NLC and the electromotility of OHC and prestin-expressing HEK. It is thought to compete with anions such as chloride for the anion-binding site on prestin. Other NSAIDs such as ibuprofen, naproxen, piroxicam or diflusinal can trigger side effects related with hearing and sometime cause tinnitus by an unknown mechanism. Here, we investigate a possible mechanism for these adverse reactions by examining the effects of these drugs on the function of prestin. The prestin-associated NLC is monitored by whole-cell as well as inside-out patches from HEK cells expressing prestin, and recorded before and after the perfusion of NSAIDs. This allows a cell-by-cell comparison of the NLC parameters in the presence and absence of the tested molecule. Ibuprofen and naproxen both showed an effect on the half-maximum voltage (V1/2) and the charge density parameters of the NLC. The effect of ibuprofen was particularly pronounced, shifting V1/2 from −70 mV to −52 mV and increasing the maximal charge movement by 30%. Further studies will contribute to our understanding of whether NSAIDs act through alteration of the mechanical properties of the membrane or specifically interfere with prestin function.