Mediation Of Prestin Expression And Piezoelectricity On Auditory Sensitivity And High Frequency Responsibility

Siting above the basllar membrane,outer hair cells(OHCs) appear able to perceive its vibration through their mechanosensitive hair bundles and to feed back mechanical forces that enhance both auditory system's sensitivity and its frsquency selectivity.They are mechanical components of the cochlea and have been the subject of many experiments designed to discover just exactly how they generate mechanical forces and how these forces contribute to the micromechanics of the cochlea.The electromotilty of the cell body has been the subject of the most extensive studies,which is a high-frequency length change of up to 5%that can be driven at acoustic frequencies and that it is not directly dependent on ATP or calcium ion but it does require the cytoplasm to have positive turgor pressure.This electromotilty is both fast enough and strong enough to sharpen the sound-induced mechanical displacements of the basilar membrane.OHC electromotility is a direct transduction of cell length change to membrane potential and is functionally equivalent to the transduction piezoelectric.Prestin is required for OHC motility and plays a central role in OHC electromotility.Today,we still have no knowledge of Prestin's most nature,such as the three dimensional organization.Without definitive information,it is difficult to understand how is alters electromechanical coupleing the OHC PM, how is involved in the mammalian cochlear amplifier. Part one:Prestin is expressed on the whole outer hair cell basolateral surfacePrestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility.Previous experiments revealed that OHC electromotility and its associated nonlinear capacitance resided in the OHC lateral wall and was not detected at the apical cuticular plate and basal region.In this experiment,the distribution of prestin in adult mouse,rat,and guinea pig OHCs was re-examined by use of immunofluorescent staining and confocal microscopy.We found that prestin labeling was located at the whole OHC basolateral wall,including the basal plasma membrane.However,staining at the basal membrane was weak.As compared with the intensity at the lateral wall,the intensities of prestin labeling at the membrane at the nuclear level and basal pole were 80.5%and 61.1%, respectively.Prestin labeling was not found at the cuticular plate and stereocilia. The prestin labeling was also absent in the cytoplasmand nuclei.The OHC lateral wall above the nuclear level is composed of the plasmamembrane,cortical lattice, and subsurface cisternae.By costaining with di-8-ANEPPS,prestin labeling was found at the outer layer of the OHC lateral wall,which was further evidenced by use of a hypotonic challenge to separate the plasma membrane from the underlying subsurface cisternae.The data revealed that prestin is expressed at the whole OHC basolateral membrane.Prestin in the basal plasma membrane may provide a reservoir on the OHC surface for prestin-recycling and may also facilitate performing its hypothesized transporter functionPart two:Prestiu up-regulation in chronic salicylate(aspirin) administration: An implication of functional dependence of prestin expressionSalicylate(aspirin) can reversibly eliminate outer hair cell(OHC) electromotility to induce hearing loss.Prestin is the OHC electromotility motor protein.Here we report that,consistence 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 fourfold.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)Ⅱexpression but down-regulated NF-kB 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.Part three:Piezoelectricity Increases Outer Hair Cell High Frequency ResponseOuter hair cell(OHC) electromotility is a cochlear amplifier and can actively boost the basilar membrane vibration to enhance auditory sensitivity and frequency selectivity.OHC electromotility is membrane-potential dependent and driven by cross-membrane voltage.Although the conformation of prestin motor proteins can be rapidly changed up to 100 kHz,its driving force(cross-membrane voltage) would be dramatically attenuated at high frequency by membrane capacitance,which forms a lowpass filter with cut-frequency less than 1 kHz. Outer hair cells also have remarkable piezoelectricity.Mechanically elongating and compressing OHC can produce electric currents.Here,we report that OHC piezoelectricity can overcome membrane capacitance damping to improve OHC high frequency responses.The OHC piezoelectric response showed a high-pass property and was increased as the stimulus frequency was increased.The cutfrequency was 70-90 kHz,mainly limited by the recording system. Simultaneous administrations of electronic and mechanical(piezoelectric) stimulation to the OHC,which mimics the OHC suffered electronic(receptor current through transduction channels) and mechanical(the vibration of the basilar membrane) stimulations in vivo,generated the flat response up to 80 kHz. Abolishment of piezoelectricity eliminated this high frequency enhancement.Like a regular cell,the sole electronic frequency response of the OHC was low-pass; the cutfrequency was～1 kHz.Finally,as computer modeling expected,the resonant peaks were also visible in the responses to electronic-mechanical stimulation.Our results indicate that OHC electromotility can perform at high frequency effectively to contribute active cochlear mechanics in whole mammalian auditory frequency range.Part four:Contribution of cochlear supporting cells and gap junctional coupling to control of hearing sensitivity in the inner earDelicious mammalian hearing function relies upon outer hair cell(OHC) electromotility to increase hearing sensitivity and frequency selectivity.In situ, OHCs are constrained by Deiters supporting cells.Here,we report that in addition to support function Deiters cells(DCs) can induce OHC movement and mediate OHC electromotility.DCs had no direct electrical conductance with OHCs. However,voltage and current stimulations in DCs could affect OHC electromotility.Depolarization of DCs reduced OHC electromotility associated nonlinear capacitance(NLC) and frequency responses.Breaking DC-OHC mechanical coupling or destroying DC cytoskeleton abolished this effect.We also found that uncoupling of gap junctional coupling between DCs could induce large changes in the membrane potential and current and shifted the NLC voltage dependence.DCs could also enhance the ATP effect on OHC electromotility. These data suggest that DCs can directly mediate OHC electromotility through the cytoskeleton to alter membrane tension.These findings also provide new understandings of hearing control and gap junctional function in the inner ear.The above results imply that basal plasma membrane of OHC may provide a reservoir on the OHC surface for prestin,Prestin's expression can be modulated. These data also suggest that DCs can directly mediate OHC electromotility through the cytoskeleton to alter membrane tension and piezoelectricity of OHC enhaces voltage changes at high frequencies that may contribute to auditory system's sensitivity and its frsquency selectivity.