Outer Hair Cell Electromotility Research Articles

αII-βV spectrin bridges the plasma membrane and cortical lattice in the lateral wall of the auditory outer hair cells

The sensitivity and frequency selectivity of the mammalian cochlea involves a mechanical amplification process called electromotility, which requires prestin-dependent length changes of the outer hair cell (OHC) lateral wall in response to changes in membrane electric potential. The cortical lattice, the highly organized cytoskeleton underlying the OHC lateral plasma membrane, is made up of F-actin and spectrin. Here, we show that alphaII and two of the five beta-spectrin subunits, betaII and betaV, are present in OHCs. betaII spectrin is restricted to the cuticular plate, a dense apical network of actin filaments, whereas betaV spectrin is concentrated at the cortical lattice. Moreover, we show that alphaII-betaV spectrin directly interacts with F-actin and band 4.1, two components of the OHC cortical lattice. betaV spectrin is progressively recruited into the cortical lattice between postnatal day 2 (P2) and P10 in the mouse, in parallel with prestin membrane insertion, which itself parallels the maturation of cell electromotility. Although betaV spectrin does not directly interact with prestin, we found that addition of lysates derived from mature auditory organs, but not from the brain or liver, enables betaV spectrin-prestin interaction. Using this assay, betaV spectrin, via its PH domain, indirectly interacts with the C-terminal cytodomain of prestin. We conclude that the cortical network involved in the sound-induced electromotility of OHCs contains alphaII-betaV spectrin, and not the conventional alphaII-betaII spectrin.

Human hereditary hearing impairment: mouse models can help to solve the puzzle

Mouse models are important research tools in the discovery of genes for syndromic as well as non-syndromic forms of hearing impairment. The similarity between the human and mouse inner ear, as well as the high homology between the human and mouse genome, make the mouse an excellent tool for genetic research on hearing loss. Over the past ten years, multiple mouse models for human hereditary hearing loss have been identified. The current review discusses the models according to the biological process in which they are involved. We review mouse models for genes involved in acellular matrix composition, hair bundle formation and maintenance, outer hair cell electromotility, the hair cell ribbon synapse and auditory nerve transmission, regulation of cochlear homeostasis and regulation of transcription. We have opted to put most emphasis on mouse models for non-syndromic forms of human hearing loss. However, we also discuss advances made in the field of age related hearing impairment by the use of mouse models and modifiers of hearing loss genes identified in mice.

Three-dimensional current flow in a large-scale model of the cochlea and the mechanism of amplification of sound.

The mammalian inner ear uses its sensory hair cells to detect and amplify incoming sound. It is unclear whether cochlear amplification arises uniquely from a voltage-dependent mechanism (electromotility) associated with outer hair cells (OHCs) or whether other mechanisms are necessary, for the voltage response of OHCs is apparently attenuated excessively by the membrane electrical filter. The cochlea contains many thousands of hair cells organized in extensive arrays, embedded in an electrically coupled system of supporting cells. We have therefore constructed a multi-element, large-scale computational model of cochlear sound transduction to study the underlying potassium (K+) recirculation. We have included experimentally determined parameters of cochlear macromechanics, which govern sound transduction, and data on hair cells' electrical parameters including tonotopical variation in the membrane conductance of OHCs. In agreement with the experiment, the model predicts an exponential decay of extracellular longitudinal K+ current spread. In contrast to the predictions from isolated cells, it also predicts low attenuation of the OHC transmembrane receptor potential (-5 dB per decade) in the 0.2-30 kHz range. This suggests that OHC electromotility could be driven by the transmembrane potential. Furthermore, the OHC electromotility could serve as a single amplification mechanism over the entire hearing range.

Membrane Composition Modulates Prestin-associated Charge Movement

The lateral membrane of the cochlear outer hair cell (OHC) is the site of a membrane-based motor that powers OHC electromotility, enabling amplification and fine-tuning of auditory signals. The OHC membrane protein prestin plays a central role in this process. We have previously shown that membrane cholesterol modulates the peak voltage of prestin-associated nonlinear capacitance in vivo and in vitro. The present study explores the effects of membrane cholesterol and docosahexaenoic acid content on the peak and magnitude of prestin-associated charge movement in a human embryonic kidney (HEK 293) cell model. Increasing membrane cholesterol results in a hyperpolarizing shift in the peak voltage of the nonlinear capacitance (Vpkc) and a decrease in the total charge movement. Both measures depend linearly on membrane cholesterol concentration. Incubation of cholesterol-loaded cells in cholesterol-free media partially restores the Vpkc toward normal values but does not have a compensatory effect on the total charge movement. Decreasing membrane cholesterol results in a depolarizing shift in Vpkc that is restored toward normal values upon incubation in cholesterol-free media. However, cholesterol depletion does not alter the magnitude of charge movement. In contrast, increasing membrane docosahexaenoic acid results in a hyperpolarizing shift in Vpkc that is accompanied by an increase in total charge movement. Our results quantify the relation between membrane cholesterol concentration and prestin-associated charge movement and enhance our understanding of how membrane composition modulates prestin function.

Prestin up-regulation in chronic salicylate (aspirin) administration: an implication of functional dependence of prestin expression.

Salicylate (aspirin) can reversibly eliminate outer hair cell (OHC) electromotility to induce hearing loss. Prestin is the OHC electromotility motor protein. Here we report that, consistency with increase in distortion product otoacoustic emission, long-term administration of salicylate can increase prestin expression and OHC electromotility. The prestin expression at the mRNA and protein levels was increased by three- to four-fold. In contrast to the acute inhibition, the OHC electromotility associated charge density was also increased by 18%. This incremental increase was reversible. After cessation of salicylate administration, the prestin expression returned to normal. We also found that long-term administration of salicylate did not alter cyclooxygenase (Cox) II expression but down-regulated NF-kappaB and increased nuclear transcription factors c-fos and egr-1. The data suggest that prestin expression in vivo is dynamically up-regulated to increase OHC electromotility in long-term administration of salicylate via the Cox-II-independent pathways.

ATP activates P2x receptors and requires extracellular Ca++ participation to modify outer hair cell nonlinear capacitance

Intracochlear ATP is an important mediator in regulating hearing function. ATP can activate ionotropic purinergic (P2x) and metabotropic purinergic (P2y) receptors to influence cell functions. In this paper, we report that ATP can activate P2x receptors directly to modify outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining hearing sensitivity and frequency selectivity in mammals. We found that ATP, but not UTP, a P2y receptor agonist, reduced the OHC electromotility-associated nonlinear capacitance (NLC) and shifted its voltage dependence to the right (depolarizing) direction. Blockage of the activation of P2x receptors by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suramin, and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) could block the ATP effect. This modification also required extracellular Ca(++) participation. Removal of extracellular Ca(++) abolished the ATP effect. However, chelation of intracellular Ca(++) concentration by a fast calcium-chelating reagent 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10 mM) did not affect the effect of ATP on NLC. The effect is also independent of K(+) ions. Substitution of Cs(+) for intracellular or extracellular K(+) did not affect the ATP effect. Our findings indicate that ATP activates P2x receptors instead of P2y receptors to modify OHC electromotility. Extracellular Ca(++) is required for this modification.

Gerbils can tune in

Gerbils can tune in

The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities

Prestin is a transmembrane motor protein localized at the outer hair cells (OHCs) of the mammalian inner ear. Voltage-dependent conformational changes in prestin generate changes in the length of OHCs. A loss of prestin function is reported to induce severe auditory deficiencies, suggesting prestin-dependent changes of OHC length may be at least a part of cochlear amplification. Here we expressed the recombinant FLAG-fused prestin proteins in Sf9 cells and purified to particles of a uniform size in EM. The square-shaped top view of purified prestin, the binding of multiple anti-FLAG antibodies to each prestin particle, the native-PAGE analysis, and the much larger molecular weight obtained from size exclusion chromatography than the estimation for the monomer all support that prestin is a tetramer (Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., and Cheatham, M. (2006) J. Biol. Chem. 281, 19916-19924). From negatively stained prestin particles, the three-dimensional structure was reconstructed at 2 nm resolution assuming 4-fold symmetry. Prestin is shown to be a bullet-shaped particle with a large cytoplasmic domain. The surface representation demonstrates indentations on the molecule, and the slice images indicate the inner cavities of sparse densities. The dimensions, 77 x 77 x 115 A, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.

Subpixel tracking for the analysis of outer hair cell movements

Conclusion. Videomicroscopy with subpixel analysis is an excellent system for quantification of outer hair cell (OHC) movements. The resolution of a few nanometers is accurate enough to show induced differences of electromotility. Objective. Electromotility of OHCs is a voltage-dependent process resulting from a membrane protein named prestin. Voltage sensitivity is conferred to prestin by intracellular anions. Reduction of these anions reduces electromotility. Videomicroscopy and subpixel tracking combine video-based analysis with a resolution of few nanometers. The aim of this study was to show the feasibility of a system for quantification of OHC movements. Materials and methods. Electromotility was investigated under normal and reduced intracellular chloride conditions. Cells were stimulated by the patch-clamp technique. Voltage steps were 500 ms long, ranging from −170 to +30 mV in 10 mV steps. Results. As in previous studies our results show the following. The direction of OHC movement depends on the polarity of voltage steps, length changes are not equal for symmetrical voltage steps of opposite polarity, average shortening for a depolarizing step (−70 mV to +30 mV) is about 13 nm/mV. Hyperpolarization (−70 mV to −170 mV) on average evokes elongations of about 3 nm/mV. Half maximal chloride concentration reduces motility by 14%; half maximal electromotility is reached by a 94% reduction of chloride.

Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin

The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505–744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529–744], PreCDL, and [529–720], PreCDS). These two polypeptides were purified to homogeneity and characterised by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The two proteins possess a three-dimensional structure and show a great tendency to aggregate. In particular, PreCDL is present in solution mainly as dimers and tetramers. These data correlate with that of full-length prestin that forms stable tetramers, suggesting that the C-terminal domain play an important role in modulating the properties of the entire prestin.

Tuning of the Outer Hair Cell Motor by Membrane Cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Absence of Voltage-Dependent Compliance in High-Frequency Cochlear Outer Hair Cells

Cochlear outer hair cells are the key element in a mechanical amplification process that enhances auditory sensitivity and tuning in the mammalian inner ear. The electromotility of outer hair cells, that is, their ability to extend or contract at acoustic frequencies, is proposed to be the source of the mechanical amplification. For amplification to take place, some stiffness is required for outer hair cells to communicate force to the organ of Corti, the sensory epithelium of the inner ear. Modulation of this stiffness would be expected to have a significant effect on inner ear function. Outer hair cell compressive stiffness has recently been shown to be dependent on membrane potential, but this has only been demonstrated for cells originating in the apical, low-frequency segment of the cochlea, whereas cochlear amplification is arguably more important in the more basal high-frequency segment. The voltage-dependent compliance (the reciprocal of stiffness) of high-frequency outer hair cells was investigated by two methods in cells isolated from the basal turns of the guinea pig cochlea. In contrast to previous findings, no evidence was found for voltage-dependent changes in compliance. The results call into question the importance of outer hair cell voltage-dependent compliance as a component of cochlear amplification.

A new mutation in the human pres gene and its effect on prestin function

The electromotility of cochlear outer hair cells (OHCs) is a major factor in cochlear amplification that enhances the sensitivity of hearing in humans. Prestin is associated with presumed conformational changes in an integral membrane protein. Prestin knockout (-/-) mice display loss of OHC electromotility and a 40- to 60-dB reduction in cochlear sensitivity in vivo. In the present study we described the results of a direct sequencing mutation in the pres gene that was found in genetic screening performed in 47 patients characterized by non-syndromic, mild-to-moderate hearing impairment (30-70 dB) and in 50 control subjects from Hungary, after exclusion of GJB (GJB2, GJB6) mutations in the background. Only one patient and his normal-hearing father showed a heterozygous missense mutation (R150Q/WT) in the 6th coding exon of the pres gene. None of the 50 control subjects with normal hearing carried this mutation. Electrophysiological studies on the R150Q (homozygous and heterozygous) prestin mutant transiently transfected into reporting cells demonstrated nonlinear capacitance functions (NLC) as a signature of OHC electromotility. The capacitance function in human kidney cell line TSA 201 was similar for wild-type prestin and the mutant. However, for the mutant the voltage where the maximal charge displacement occurred (V1/2) significantly shifted in the hyperpolarizing direction ( approximately 15 mV). This is the first genetic and electrophysiological analysis of a human mutation in a coding exon of the pres gene by 47 patients with non-syndromic, sensorineural, mild-to-moderate hearing impairment; although the pathogenic role of the R150Q mutation is not unambiguous.

Streptomycin and gentamicin have no immediate effect on outer hair cell electromotility

The cochlear outer hair cell (OHC), which plays a crucial role in mammalian hearing through its unique voltage-dependent motility, has been established as a primary target of the ototoxicity of aminoglycoside antibiotics. These polycationic drugs are also known to block a wide variety of ion channels, purinergic ionotropic channels, and nicotinic ACh receptors in hair cells in vitro. The OHC motor protein, prestin, is a voltage-sensitive transmembrane protein containing several negatively charged residues on both intra- and extracellular surface. The acidic sites may be susceptible to polycationic-charged aminoglycoside binding, which may result in disruption of motility. We attempted to examine whether aminoglycosides such as streptomycin and gentamicin could affect OHC motility and its electrical signature, the nonlinear capacitance (NLC) in adult gerbil OHCs. Somatic motility and NLC were measured under the whole-cell voltage-clamp mode. Streptomycin and gentamicin were applied extracellularly or intracellularly. Results show that streptomycin and gentamicin did not change either the magnitude of motility or the NLC. Theses results suggest that, although streptomycin and gentamicin can block mechanotransduction channels as well as ACh receptors in hair cells, they have no direct affect on OHC somatic motility.

Prestin-based outer hair cell electromotility in knockin mice does not appear to adjust the operating point of a cilia-based amplifier

The remarkable sensitivity and frequency selectivity of the mammalian cochlea is attributed to a unique amplification process that resides in outer hair cells (OHCs). Although the mammalian-specific somatic motility is considered a substrate of cochlear amplification, it has also been proposed that somatic motility in mammals simply acts as an operating-point adjustment for the ubiquitous stereocilia-based amplifier. To address this issue, we created a mouse model in which a mutation (C1) was introduced into the OHC motor protein prestin, based on previous results in transfected cells. In C1/C1 knockin mice, localization of C1-prestin, as well as the length and number of OHCs, were all normal. In OHCs isolated from C1/C1 mice, nonlinear capacitance and somatic motility were both shifted toward hyperpolarization, so that, compared with WT controls, the amplitude of cycle-by-cycle (alternating, or AC) somatic motility remained the same, but the unidirectional (DC) component reversed polarity near the OHC's presumed in vivo resting membrane potential. No physiological defects in cochlear sensitivity or frequency selectivity were detected in C1/C1 or C1/+ mice. Hence, our results do not support the idea that OHC somatic motility adjusts the operating point of a stereocilia-based amplifier. However, they are consistent with the notion that the AC component of OHC somatic motility plays a dominant role in mammalian cochlear amplification.

Role of lipids in electromotility of outer hair cells

Role of lipids in electromotility of outer hair cells

Voltage‐sensitive prestin orthologue expressed in zebrafish hair cells

Prestin, a member of the solute carrier (SLC) family SLC26A, is the molecular motor that drives the somatic electromotility of mammalian outer hair cells (OHCs). Its closest reported homologue, zebrafish prestin (zprestin), shares approximately 70% strong amino acid sequence similarity with mammalian prestin, predicting an almost identical protein structure. Immunohistochemical analysis now shows that zprestin is expressed in hair cells of the zebrafish ear. Similar to mammalian prestin, heterologously expressed zprestin is found to generate voltage-dependent charge movements, giving rise to a non-linear capacitance (NLC) of the cell membrane. Compared with mammalian prestin, charge movements mediated by zprestin display a weaker voltage dependence and slower kinetics; they occur at more positive membrane voltages, and are not associated with electromotile responses. Given this functional dissociation of NLC and electromotility and the structural similarity with mammalian prestin, we anticipate that zprestin provides a valuable tool for tracing the molecular and evolutionary bases of prestin motor function.

Prestin–prestin and prestin–GLUT5 interactions in HEK293T cells

The remarkable hearing sensitivity and frequency selectivity in mammals is attributed to cochlear amplifier in the outer hair cells (OHCs). Prestin, a membrane protein in the lateral wall of OHC plasma membrane, is required for OHC electromotility and cochlear amplifier. In addition, GLUT5, a fructose transporter, is reported to be abundant in the plasma membrane of the OHC lateral wall and has been originally proposed as the OHC motor protein. Here we provide evidence of interactions between prestin/prestin and prestin/GLUT5 in transiently transfected HEK293T cells. We used a combination of techniques: (1) membrane colocalization by confocal microscopy, (2) fluorescence resonance energy transfer (FRET) by fluorescence activated cell sorting (FACS), (3) FRET by acceptor photobleaching, (4) FRET by fluorescence lifetime imaging (FRET-FLIM), and (5) coimmunoprecipitation. Our results suggest that homomeric and heteromeric prestin interactions occur in native OHCs to facilitate its electromotile function and that GLUT5 interacts with prestin for its elusive function.

Overexpression of SK2 Channels Enhances Efferent Suppression of Cochlear Responses Without Enhancing Noise Resistance

Cochlear hair cells express SK2, a small-conductance Ca(2+)-activated K(+) channel thought to act in concert with Ca(2+)-permeable nicotinic acetylcholine receptors (nAChRs) alpha9 and alpha10 in mediating suppressive effects of the olivocochlear efferent innervation. To probe the in vivo role of SK2 channels in hearing, we examined gene expression, cochlear function, efferent suppression, and noise vulnerability in mice overexpressing SK2 channels. Cochlear thresholds, as measured by auditory brain stem responses and otoacoustic emissions, were normal in overexpressers as was overall cochlear morphology and the size, number, and distribution of efferent terminals on outer hair cells. Cochlear expression levels of SK2 channels were elevated eightfold without striking changes in other SK channels or in the alpha9/alpha10 nAChRs. Shock-evoked efferent suppression of cochlear responses was significantly enhanced in overexpresser mice as seen previously in alpha9 overexpresser mice; however, in contrast to alpha9 overexpressers, SK2 overexpressers were not protected from acoustic injury. Results suggest that efferent-mediated cochlear protection is mediated by other downstream effects of ACh-mediated Ca(2+) entry different from those involving SK2-mediated hyperpolarization and the associated reduction in outer hair cell electromotility.

Application of fluorescence polarization microscopy to measure fluorophore orientation in the outer hair cell plasma membrane

The biophysical properties and organization of cell membranes regulate many membrane-based processes, including electromotility in outer hair cells (OHCs) of the cochlea. Studies of the membrane environment can be carried out by measuring the orientation of membrane-bound fluorophores using fluorescence polarization microscopy (FPM). Due to the cylindrical shape of OHCs, existing FPM theory developed for spherical cells is not applicable. We develop a new method for analyzing FPM data suitable for the quasi-cylindrical OHC. We present the theory for this model, as well as a study of the orientation of the fluorescent probe pyridinium, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl) (di-8-ANEPPS) in the OHC membrane. Our results indicate that the absorption transition dipole moment of di-8-ANEPPS orients symmetrically about the membrane normal at 27 deg with respect to the plane of the membrane. The observed agreement between theoretical predictions and experimental measurements establishes the applicability of FPM to study OHC plasma membrane properties.