An anion antiporter model of prestin, the outer hair cell motor protein

Cochlear amplification in mammalian hearing relies on an active mechanical feedback process generated by outer hair cells, driven by a protein, prestin (SLC26A5), in the lateral membrane. We have used kinetic models to understand the mechanism by which prestin might function. We show that the two previous hypotheses of prestin, which assume prestin cannot operate as a transporter, are insufficient to explain previously published data. We propose an alternative model of prestin as an electrogenic anion exchanger, exchanging one Cl(-) ion for one divalent or two monovalent anions. This model can reproduce the key aspects of previous experimental observations. The experimentally observed charge movements are produced by the translocation of one Cl(-) ion combined with intrinsic positively charged residues, while the transport of the counteranion is electroneutral. We tested the model with measurements of the Cl(-) dependence of charge movement, using SO(4)(2-) to replace Cl(-). The data was compatible with the predictions of the model, suggesting that prestin does indeed function as a transporter.     

Tension sensitivity of prestin: comparison with the membrane motor in outer hair cells

The membrane motor in outer hair cells undergoes conformational transitions involving charge displacement of approximately 0.8 e across the membrane and changes of approximately 4 nm(2) in its membrane area. Previous reports have established that the charge transfer in the membrane motor and that in prestin, a membrane protein in the plasma membrane of outer hair cells, are approximately equal. Here, we determine the membrane area changes based on its sensitivity to membrane tension. We found that prestin does undergo area changes and that the magnitude is approximately 1 nm(2), smaller than the value 4 nm(2) for outer hair cell motor. This result confirms that prestin is a protein that functions as a membrane motor based on piezoelectricity. The discrepancy in the magnitude could suggest a prestin-containing complex in outer hair cells.     

哺乳动物耳蜗外毛细胞的马达蛋白:Prestin

近几年的研究发现,在耳蜗基底膜的外毛细胞膜上有一种新奇的蛋白质:prestin(马达蛋白),它能感受细胞膜电位的变化,进而发生构象改变,引发外毛细胞的形状和表面积的改变.Prestin作为一种独特的马达蛋白,能驱动耳蜗外毛细胞的电能动性(electromotility),产生耳蜗的放大器作用,因而使哺乳动物的听觉具有高度的敏感性,广阔的听觉域,敏锐的频率选择性.这种蛋白质的缺失或基因的突变会导致听觉功能严重受损,对于prestin的深入细致的研究,也许可以使人们进一步认识和理解哺乳动物的听觉调谐机制,通过对这种蛋白质基因的表达的调控,是否能够防治一些与之相关的疾病?这或许将是今后听觉研究领域的一个重要课题.     

A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity

Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.     

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.     

Analysis of the oligomeric structure of the motor protein prestin

Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed approximately 11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an approximately 11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestin-prey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the approximately 11-nM particles in the outer hair cell's basolateral membrane.     

Effects of chlorpromazine and trinitrophenol on the membrane motor of outer hair cells

The motile activity of outer hair cells' cell body is associated with large nonlinear capacitance due to a membrane motor that couples electric displacement with changes in the membrane area, analogous to piezoelectricity. This motor is based on prestin, a member of the SLC26 family of anion transporters and utilizes the electric energy available at the plasma membrane associated with the sensory function of these cells. To understand detailed mechanism of this motile activity, we examined the effect of amphipathic ions, cationic chlorpromazine and anionic trinitrophenol, which are thought to change the curvature of the membrane in opposite directions. We found that both chemicals reduced cell length at the holding potential of -75 mV and induced positive shifts in the cells' voltage dependence. The shift observed was approximately 10 mV for 500 microM trinitrophenol and 20 mV for 100 microM cationic chlorpromazine. Length reduction at the holding potential and voltage shifts of the motile activity were well correlated. The voltage shifts of nonlinear capacitance were not diminished by eliminating the cells' turgor pressure or by digesting the cortical cytoskeleton. These observations suggest that the membrane motor undergoes conformational transitions that involve changes not only in membrane area but also in bending stiffness.     

Mapping the distribution of the outer hair cell motility voltage sensor by electrical amputation.

The outer hair cell (OHC) possesses a nonlinear charge movement whose characteristics indicate that it represents the voltage sensor responsible for OHC mechanical activity. OHC mechanical activity is known to exist along a restricted extent of the cell's length. We have used a simultaneous partitioning microchamber and whole cell voltage clamp technique to electrically isolate sections of the OHC membrane and find that the nonlinear charge movement is also restricted along the cell's length. Apical and basal portions of the OHC are devoid of voltage sensors, corresponding to regions of the cell where the subsurface cisternae and/or the mechanical responses are absent. We conclude that the physical domain of the motility voltage sensor corresponds to that of the mechanical effector and speculate that sensor and effector reside within one intra membranous molecular species, perhaps an evolved nonconducting or poorly conducting voltage-dependent ion channel.     

Piezoelectric reciprocal relationship of the membrane motor in the cochlear outer hair cell

It has been shown that the membrane motor in the outer hair cell is driven by the membrane potential. Here we examine whether the motility satisfies the reciprocal relationship, the characteristic of piezoelectricity, by measuring charge displacement induced by stretching the cell with known force. The efficiency of inducing charge displacement was membrane potential dependent. The maximum efficiency of inducing charge displacement by force was approximately 20 fC/nN for 50-microm-long lateral membrane. The efficiency per cell stretching was 0.1 pC/microm. We found that these values are consistent with the reciprocal relationship based on the voltage sensitivity of approximately 20 nm/mV for 50-microm-long cell and force production of 0.1 nN/mV by the cell. We can thus conclude that the membrane motor in the outer hair cell satisfies a necessary condition for piezoelectricity and that the hair cell's piezoelectric coefficient of 20 fC/nN is four orders of magnitude greater than the best man-made material.     

Fluctuation of motor charge in the lateral membrane of the cochlear outer hair cell

Functioning of the membrane motor of the outer hair cell is tightly associated with transfer of charge across the membrane. To obtain further insights into the motor mechanism, we examined kinetics of charge transfer across the membrane in two different modes. One is to monitor charge transfer induced by changes in the membrane potential as an excess membrane capacitance. The other is to measure spontaneous flip-flops of charges across the membrane under voltage-clamp conditions as current noise. The noise spectrum of current was inverse Lorentzian, and the capacitance was Lorentzian, as theoretically expected. The characteristic frequency of the capacitance was approximately 10 kHz, and that for current noise was approximately 30 kHz. The difference in the characteristic frequencies seems to reflect the difference in the modes of mechanical movement associated with the two physical quantities.     

Evidence of piezoelectric resonance in isolated outer hair cells

Our results demonstrate high-frequency electrical resonances in outer hair cells (OHCs) exhibiting features analogous to classical piezoelectric transducers. The fundamental (first) resonance frequency averaged f(n) approximately 13 kHz (Q approximately 1.7). Higher-order resonances were also observed. To obtain these results, OHCs were positioned in a custom microchamber and subjected to stimulating electric fields along the axis of the cell (1-100 kHz, 4-16 mV/80 microm). Electrodes embedded in the side walls of the microchamber were used in a voltage-divider configuration to estimate the electrical admittance of the top portion of the cell-loaded chamber (containing the electromotile lateral wall) relative to the lower portion (containing the basal plasma membrane). This ratio exhibited resonance-like electrical tuning. Resonance was also detected independently using a secondary 1-MHz radio-frequency interrogation signal applied transversely across the cell diameter. The radio-frequency interrogation revealed changes in the transverse electric impedance modulated by the axial stimulus. Modulation of the transverse electric impedance was particularly pronounced near the resonant frequencies. OHCs used in our study were isolated from the apical region of the guinea pig cochlea, a region that responds exclusively to low-frequency acoustic stimuli. In this sense, electrical resonances we observed in vitro were at least an order of magnitude higher (ultrasonic) than the best physiological frequency of the same OHCs under acoustic stimuli in vivo. These resonance data further support the piezoelectric theory of OHC function, and implicate piezoelectricity in the broad-band electromechanical behavior of OHCs underlying mammalian cochlear function.     

Structural features of the lateral walls in mammalian cochlear outer hair cells

Summary Freeze-fracture, freeze-etching and thin sections have been used to determine features of the structural organisation of the lateral walls in cochlear outer hair cells. The presence of an organised meshwork of filaments in the lateral cortex of the cell is confirmed in intact unfixed cells. This meshwork showed morphological features similar to the cytoskeletal lattice. The lateral plasma membrane is shown to be protein-rich and to contain cholesterol. The membranes of the subplasmalemmal lateral cisternae contain much less protein, and little cholesterol as judged by their responses to filipin and tomatin. These findings indicate differences in the physical properties of the two membrane systems. On the fracture faces of the plasma membrane there is a high density of intramembrane particles and this particle population is heterogeneous. Some particles show morphological features consistent with those of transmembrane channels. Regularly spaced pillars crossing the space between the plasma and cisternal membranes were identified both in thin sections and in freezeetched preparations, but neither the plasma nor cisternal membrane fracture faces showed any feature corresponding directly to the pillar. This suggests the pillars do not insert directly into either membrane. Freeze-fracture and freeze-etching of unfixed cells indicated that the pillar is indirectly associated with the cytoplasmic surface of the plasma membrane, and, at its inner end, linked to the cortical cytoskeletal lattice on the outer surface of the cisternal membrane.     

ROCK-dependent and ROCK-independent control of cochlear outer hair cell electromotility

Outer hair cell electromotility is crucial for the proper function of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear. Previous work (Kalinec, F., Zhang, M., Urrutia, R., and Kalinec, G. (2000) J. Biol. Chem. 275, 28000-28005) has suggested a role for Rho GTPases in the regulation of outer hair cell electromotility, although the signaling pathways mediated by these enzymes remain to be established. Here we have investigated the cellular and molecular mechanisms underlying the homeostatic regulation of the electromotile response of guinea pig outer hair cells. Our findings defined a ROCK-mediated signaling cascade that continuously modulates outer hair cell electromotility by selectively targeting the cytoskeleton. A distinct ROCK-independent pathway functions as a fast resetting mechanism for this system. Neither pathway affects the function of prestin, the unique molecular motor of outer hair cells. These results extend our understanding of a basic mechanism of both normal human hearing and deafness, revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility and suggesting ROCK as a molecular target for modulating the function of the cochlear amplifier.     

Atomic force microscopy imaging of the structure of the motor protein prestin reconstituted into an artificial lipid bilayer

Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti-prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring-like with a diameter of about 11 nm.     

High frequency force generation in outer hair cells from the mammalian ear

Mammalian outer hair cells generate mechanical forces at acoustic frequencies and can thus amplify the sound stimulus within the inner ear. The mechanism of force generation depends upon the plasma membrane potential but not upon either calcium or ATP. Forces are generated in the lateral cortex along the full length of the cell. The cortex includes a two-dimensional cytoskeletal lattice composed of circumferential filaments 6-7 nm thick that are cross-linked by filaments 3-4 nm thick and 40-60 nm long. The two filament types may, respectively, be actin and some form of spectrin. The lattice reinforces the cylindrical shape of the cell and permits limited changes in length. Beneath it lie the lateral cisternae, a regular system of multi-layered membranes. Force-generation may depend upon voltage-dependent shape changes in proteins that lie either in the plasma membrane or in the cytoskeletal lattice.     

Effect of membrane mechanics on charge transfer by the membrane protein prestin

Prestin was found in the membrane of outer hair cells (OHCs) located in the cochlea of the mammalian inner ear. These cells convert changes in the membrane potential into dimensional changes and (if constrained) to an active electromechanical force. The OHCs provide the ear with the mechanism of amplification and frequency selectivity that is effective up to tens of kHz. Prestin is a crucial part of the motor complex driving OHCs. Other cells transfected with prestin acquire electromechanical properties similar to those in the native cell. While the mechanism of prestin has yet to be fully understood, the charge transfer is its critical component. Here we investigate the effect of the mechanics of the surrounding membrane on electric charge transfer by prestin. We simulate changes in the membrane mechanics via the corresponding changes in the free energy of the prestin system. The free energy gradient enters a Fokker-Planck equation that describes charge transfer in our model. We analyze the effects of changes in the membrane tension and membrane elastic moduli. In the case of OHC, we simulate changes in the longitudinal and/or circumferential stiffness of the cell’s orthotropic composite membrane. In the case of cells transfected with prestin, we vary the membrane areal modulus. As a result, we show the effects of the membrane mechanics on the probabilistic characteristics of prestin-associated charge transfer for both stationary and high-frequency conditions. We compare our computational results with the available experimental data and find good agreement with the experiment.     

Cholinergic control of membrane conductance and intracellular free Ca2+ in outer hair cells of the guinea pig cochlea

We have studied the action of cholinergic agonists on outer hair cells, both in situ and isolated from the cochlea of the guinea pig, combining new fast CCD technology for Ca2+ imaging and conventional patch-clamp methods. Carbachol (1 mM) activated a current with a reversal potential near -70 mV and a bell-shaped I-V curve, suggesting that it was a Ca2+ activated K+ current. In a few cells, this current was preceded by a transient inward current, probably owing to an influx of Ca2+ and other cations through the acetylcholine (ACh) receptors. The amplitude of the Ca2+ signal was maximal in a circumscribed region at the basal pole of the cell and decreased steeply towards the apical pole, compatible with Ca2+ influx and/or Ca2+ induced Ca2+ release at the cells base. The time course of the Ca2+ rise was fastest at the base, but it was still slightly slower, and more rounded, than that of the K+ current. In some recordings the K+ current was observed without any measurable change of intracellular Ca2+. The K+ current was potentiated (18%) by caffeine (5 mM), and decreased (19%) by ryanodine (0.1 mM) in the majority of cells tested. The results are discussed in terms of a labile intracellular Ca2+ store located at the base of the cell, close to the Ca2+ permeable ACh receptor channels and Ca2+ activated K+ channels, whose contribution to the Ca2+ rise occurring in the region of the channels is variable, and probably dependent on its ability to refill with Ca2+.     

Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.