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.     

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.     

Modeling the mechanics of tethers pulled from the cochlear outer hair cell membrane

Cell membrane tethers are formed naturally (e.g., in leukocyte rolling) and experimentally to probe membrane properties. In cochlear outer hair cells, the plasma membrane is part of the trilayer lateral wall, where the membrane is attached to the cytoskeleton by a system of radial pillars. The mechanics of these cells is important to the sound amplification and frequency selectivity of the ear. We present a modeling study to simulate the membrane deflection, bending, and interaction with the cytoskeleton in the outer hair cell tether pulling experiment. In our analysis, three regions of the membrane are considered: the body of a cylindrical tether, the area where the membrane is attached and interacts with the cytoskeleton, and the transition region between the two. By using a computational method, we found the shape of the membrane in all three regions over a range of tether lengths and forces observed in experiments. We also analyze the effects of biophysical properties of the membrane, including the bending modulus and the forces of the membrane adhesion to the cytoskeleton. The model's results provide a better understanding of the mechanics of tethers pulled from cell membranes.     

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.     

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.     

Prestin-driven cochlear amplification is not limited by the outer hair cell membrane time constant

Outer hair cells (OHCs) provide amplification in the mammalian cochlea using somatic force generation underpinned by voltage-dependent conformational changes of the motor protein prestin. However, prestin must be gated by changes in membrane potential on a cycle-by-cycle basis and the periodic component of the receptor potential may be greatly attenuated by low-pass filtering due to the OHC time constant (τ(m)), questioning the functional relevance of this mechanism. Here, we measured τ(m) from OHCs with a range of characteristic frequencies (CF) and found that, at physiological endolymphatic calcium concentrations, approximately half of the mechanotransducer (MT) channels are opened at rest, depolarizing the membrane potential to near -40 mV. The depolarized resting potential activates a voltage-dependent K+ conductance, thus minimizing τ(m) and expanding the membrane filter so there is little receptor potential attenuation at the cell's CF. These data suggest that minimal τ(m) filtering in vivo ensures optimal activation of prestin.     

Cytoplasmic actin and cochlear outer hair cell motility

Summary Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaing cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.     

Simulation of motor-driven cochlear outer hair cell electromotility.

We propose a three-dimensional (3D) model to simulate outer hair cell electromotility. In our model, the major components of the composite cell wall are explicitly represented. We simulate the activity of the particles/motor complexes in the plasma membrane by generating active strains inside them and compute the overall response of the cell. We also consider the constrained wall and compute the generated active force. We estimate the parameters of our model by matching the predicted longitudinal and circumferential electromotile strains with those observed in the microchamber experiment. In addition, we match the earlier estimated values of the active force and cell wall stiffness. The computed electromotile strains in the plasma membrane and other components of the wall are in agreement with experimental observations in trypsinized cells and in nonmotile cells transfected with Prestin. We discover several features of the 3D mechanism of outer hair cell electromotilty. Because of the constraints under which the motors operate, the motor-related strains have to be 2-3 times larger than the observable strains. The motor density has a strong effect on the electromotile strain. Such effect on the active force is significantly lower because of the interplay between the active and passive properties of the cell wall.     

Micropipette aspiration on the outer hair cell lateral wall

The mechanical properties of the lateral wall of the guinea pig cochlear outer hair cell were studied using the micropipette aspiration technique. A fire-polished micropipette with an inner diameter of approximately 4 microm was brought into contact with the lateral wall and negative pressure was applied. The resulting deformation of the lateral wall was recorded on videotape and subjected to morphometric analysis. The relation between the length of the aspirated portion of the cell and aspiration pressure is characterized by the stiffness parameter, K(s) = 1.07 +/- 0.24 (SD) dyn/cm (n = 14). Values of K(s) do not correlate with the original cell length, which ranges from 29 to 74 microm. Theoretical analysis based on elastic shell theory applied to the experimental data yields an estimate of the effective elastic shear modulus, mu = 15.4 +/- 3.3 dyn/cm. These data were obtained at subcritical aspiration pressures, typically less than 10 cm H2O. After reaching a critical (vesiculation) pressure, the cytoplasmic membrane appeared to separate from the underlying structures, a vesicle with a length of 10-20 microm was formed, and the cytoplasmic membrane resealed. This vesiculation process was repeated until a cell-specific limit was reached and no more vesicles were formed. Over 20 vesicles were formed from the longest cells in the experiment.     

The role of outer hair cell motility in cochlear tuning.

The mammalian cochlea's remarkable sensitivity and frequency selectivity are thought to be mediated by the mechanical feedback action of outer hair cells. New tools for measuring the movement of cochlear elements, and recent advances in modeling are increasing our knowledge of cochlear mechanics.     

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.     

Modes and balance of energy in the piezoelectric cochlear outer hair cell wall

Here, we analyze energy transformations in the outer hair cell and its effectiveness as a piezoelectric-type actuator in the cochlea. The major modes of energy are introduced, and a method to estimate the coefficients of their tension-dependence is proposed. Next, we derive balance of the mechanical and electrical parts of energy, and show two forms of the active energy associated with the motors driving electromotility. The two forms of the active energy, stored mechanical energy, and external electrical work are then introduced as functions of voltage and applied force. We use the energy balance to introduce and estimate the effectiveness of the cell's electromotile response.     

Current noise spectrum and capacitance due to the membrane motor of the outer hair cell: theory

The voltage-dependent motility of the outer hair cell is based on a membrane motor densely distributed in the lateral membrane. The gating charge of the membrane motor is manifested as a bell-shaped membrane potential dependence of the membrane capacitance. In this paper it is shown that movements of the gating charge should produce a high-pass current noise described by an inverse Lorentzian similar to the one shown by Kolb and Läuger for ion carriers. The frequency dependence of the voltage-dependent capacitance is also derived. These derivations are based on membrane motor models with two or three states. These two models lead to similar predictions on the capacitance and current noise. It is expected that the examination of the spectral properties of these quantities would be a useful means of determining the relaxation time for conformational transitions of the membrane motor.     

Adaptive evolution in mammalian proteins involved in cochlear outer hair cell electromotility

Somatic electromotility in cochlear outer hair cells, as the basis for cochlear amplification, is a mammalian novelty and it is largely dependent upon rapid cell length changes proposed to be mediated by the motor-protein prestin, a member of the solute carrier anion-transport family 26. Thus, one might predict that prestin has specifically evolved in mammals to support this unique mammalian adaptation. Using codon-based likelihood models we found evidences for positive selection in the motor-protein prestin only in the mammalian lineage, supporting the hypothesis that lineage-specific adaptation-driven molecular changes endowed prestin with the ability to mediate somatic electromotility. Moreover, signatures of positive selection were found on the alpha10, but not the alpha9, nicotinic cholinergic receptor subunits. An alpha9alpha10-containing nicotinic cholinergic receptor mediates inhibitory olivocochlear efferent effects on hair cells across vertebrates. Our results suggest that evolution-driven modifications of the alpha10 subunit probably allowed the alpha9alpha10 heteromeric receptor to serve a differential function in the mammalian cochlea. Thus, we describe for the first time at the molecular level signatures of adaptive evolution in two outer hair cell proteins only in the lineage leading to mammals. This finding is most likely related with the roles these proteins play in somatic electromotility and/or its fine tuning.     

Tonotopic relationships reveal the charge density varies along the lateral wall of outer hair cells

Outer hair cells amplify and improve the frequency selectivity of sound within the mammalian cochlea through a sound-evoked receptor potential that induces an electromechanical response in their lateral wall membrane. We experimentally show that the membrane area and linear membrane capacitance of outer hair cells increases exponentially with the electrically evoked voltage-dependent charge movement (Q(T)) and peak membrane capacitance (C(peak)). We determine the size of the different functional regions (e.g., lateral wall, synaptic basal pole) of the polarized cells from the tonotopic relationships. We then establish that Q(T) and C(peak) increase with the logarithm of the lateral wall area (A(LW)) and determine from the functions that the charge (σ(LW,) pC/μm(2)) and peak (ρ(LW,) pF/μm(2)) densities vary inversely with A(LW) (σ(LW) = 1.3/A(LW) and ρ(LW) = 9/A(LW)). This shows contrary to conventional wisdom that σ(LW) and ρ(LW) are not constant along the length of an individual outer hair cell.     

Excess plasma membrane and effects of ionic amphipaths on mechanics of outer hair cell lateral wall

The interaction between the outer hair cell (OHC) lateral wall plasma membrane and the underlying cortical lattice was examined by a morphometric analysis of cell images during cell deformation. Vesiculation of the plasma membrane was produced by micropipette aspiration in control cells and cells exposed to ionic amphipaths that alter membrane mechanics. An increase of total cell and vesicle surface area suggests that the plasma membrane possesses a membrane reservoir. Chlorpromazine (CPZ) decreased the pressure required for vesiculation, whereas salicylate (Sal) had no effect. The time required for vesiculation was decreased by CPZ, indicating that CPZ decreases the energy barrier required for vesiculation. An increase in total volume is observed during micropipette aspiration. A deformation-induced increase in hydraulic conductivity is also seen in response to micropipette-applied fluid jet deformation of the lateral wall. Application of CPZ and/or Sal decreased this strain-induced hydraulic conductivity. The impact of ionic amphipaths on OHC plasma membrane and lateral wall mechanics may contribute to their effects on OHC electromotility and hearing.     

Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification

It is a central tenet of cochlear neurobiology that mammalian ears rely on a local, mechanical amplification process for their high sensitivity and sharp frequency selectivity. While it is generally agreed that outer hair cells provide the amplification, two mechanisms have been proposed: stereociliary motility and somatic motility. The latter is driven by the motor protein prestin. Electrophysiological phenotyping of a prestin knockout mouse intimated that somatic motility is the amplifier. However, outer hair cells of knockout mice have significantly altered mechanical properties, making this mouse model unsatisfactory. Here, we study a mouse model without alteration to outer hair cell and organ of Corti mechanics or to mechanoelectric transduction, but with diminished prestin function. These animals have knockout-like behavior, demonstrating that prestin-based electromotility is required for cochlear amplification.     

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.     

N-terminal-mediated homomultimerization of prestin, the outer hair cell motor protein

The outer hair cell lateral membrane motor, prestin, drives the cell's mechanical response that underpins mammalian cochlear amplification. Little is known about the protein's structure-function relations. Here we provide evidence that prestin is a 10-transmembrane domain protein whose membrane topology differs from that of previous models. We also present evidence that both intracellular termini of prestin are required for normal voltage sensing, with short truncations of either terminal resulting in absent or modified activity despite quantitative findings of normal membrane targeting. Finally, we show with fluorescence resonance energy transfer that prestin-prestin interactions are dependent on an intact N-terminus, suggesting that this terminus is important for homo-oligomerization of prestin. These domains, which we have perturbed, likely contribute to allosteric modulation of prestin via interactions among prestin molecules or possibly between prestin and other proteins, as well.