Mapping the distribution of outer hair cell voltage-dependent conductances by electrical amputation.

The mammalian outer hair cell (OHC) functions not only as sensory receptor, but also as mechanical effector; this unique union is believed to enhance our ability to discriminate among acoustic frequencies, especially in the kilohertz range. An electrical technique designed to isolate restricted portions of the plasma membrane was used to map the distribution of voltage-dependent conductances along the cylindrical extent of the cell. We show that three voltage-dependent currents, outward K, I(K,n), and I(Ca) are localized to the basal, synaptic pole of the OHC. Previously we showed that the lateral membrane of the OHC harbors a dense population of voltage sensor-motor elements responsible for OHC motility. This segregation of membrane molecules may have important implications for auditory function. The distribution of OHC conductances will influence the cable properties of the cell, thereby potentially controlling the voltage magnitudes experienced by the motility voltage sensors in the lateral membrane, and thus the output of the "cochlear amplifier."     

A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin’s voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein’s ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin’s unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells’ role as a cochlear amplifier.     

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole- cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl >> Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume- sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)     

On the effect of prestin on the electrical breakdown of cell membranes

The voltage-dependent activity of prestin, the outer hair cell (OHC) motor protein essential for its electromotility, enhances the mammalian inner ear's auditory sensitivity. We investigated the effect of prestin's activity on the plasma membrane's (PM) susceptibility to electroporation (EP) via cell-attached patch-clamping. Guinea pig OHCs, TSA201 cells, and prestin-transfected TSA cells were subjected to incremental 50 mus and/or 50 ms voltage pulse trains, or ramps, at rates from 10 V/s to 1 kV/s, to a maximum transmembrane potential of +/-1000 mV. EP was determined by an increase in capacitance to whole-cell levels. OHCs were probed at the prestin-rich lateral PM or prestin-devoid basal portion; TSA cells were patched at random points. OHCs were consistently electroporated with 50 ms pulses, with significant resistance to depolarizing pulses. Although EP rarely occurred with 50 mus pulses, prior stimulation with this protocol had a significant effect on the sensitivity to EP with 50 ms pulses, regardless of polarity or PM domain. Consistent with these results, resistance to EP with depolarizing 10-V/s ramps was also found. Our findings with TSA cells were comparable, showing resistance to EP with both depolarizing 50-ms pulses and 10 V/s ramps. We conclude prestin significantly affects susceptibility to EP, possibly via known biophysical influences on specific membrane capacitance and/or membrane stiffness.     

Distortion Component Analysis of Outer Hair Cell Motility-Related Gating Charge

The underlying Boltzmann characteristics of motility-related gating currents of the outer hair cell (OHC) are predicted to generate distortion components in response to sinusoidal transmembrane voltages. We studied this distortion since it reflects the mechanical activity of the cell that may contribute to peripheral auditory system distortion. Distortion components in the OHC electrical response were analyzed using the whole-cell voltage clamp technique, under conditions where ionic conductances were blocked. Single or double-sinusoidal transmembrane voltage stimulation was delivered at various holding voltages, and distortion components of the current responses were detected by Fourier analysis. Current response magnitude and phase of each distortion component as a function of membrane potential were compared with characteristics of the voltage-dependent capacitance, obtained by voltage stair-step transient analysis or dual-frequency admittance analysis. The sum distortion was most prominent among the distortion components at all holding voltages. Notches in the sum (f1+f2), difference (f2−f1) and second harmonic (2f) components occur at the voltage where peak voltage-dependent capacitance resides (V _pkCm ). Rapid phase reversals also occurred at V _pkCm , but phase remained fairly stable at more depolarized and hyperpolarized potentials. Thus, it is possible to extract Boltzmann parameters of the motility-related charge movement from these distortion components. In fact, we have developed a technique to follow changes in the voltage dependence of OHC motility and charge movement by tracking the voltage at phase reversal of the f2−f1 product. When intracellular turgor pressure was changed, V _pkCm and distortion notch voltages shifted in the same direction. These data have important implications for understanding cochlear nonlinearity, and more generally, indicate the usefulness of distortion analysis to study displacement currents. Received: 31 December 1998/Revised: 12 March 1999     

Voltage clamping with single microelectrodes: comparison of the discontinuous mode and continuous mode using the Axoclamp 2A amplifier

The voltage clamp technique is a powerful method for studying the physiology of excitable membrane. This technique has made possible the determination of ionic responses generated by activation of either receptor-mediated or voltage-dependent processes. The development of the whole-cell, 'tight-seal' voltage clamp method has allowed the analysis and examination of membrane physiology at the single cell level. The method allows the characterization of voltage-dependent ionic conductances both at the macroscopic (whole-cell) and at the microscopic (unitary conductance or single channel) level in cells less than 10 micron in diameter, a feat difficult to achieve with 'conventional' fine-tipped micropipettes. In this paper, several methologies used for culturing neuronal and non-neuronal cells in the laboratory are described. A comparison between the two modes of voltage clamp using blunt-tipped 'patch'-microelectrodes, the switching (discontinuous) and the non-switching (continuous) modes, of the Axoclamp-2A amplifier is made. Some results on membrane currents obtained from neuronal and non-neuronal cells using the single electrode whole-cell 'tight-seal' voltage clamp is illustrated. The possible existence of two inactivating K+ currents, one dependent on Ca++ the other is not, is discussed.     

Potassium channel gating in adhesion: from an oocyte–silicon to a neuron–astrocyte adhesion contact

In a neuron–astrocyte adhesion contact the ionic current due to the opening of voltage-dependent potassium channels has to flow along a narrow intercellular cleft, generating there an extracellular voltage. This voltage might be large enough to affect significantly the dependence of channel gating from the intracellular voltage. In order to test this hypothesis, we considered a Xenopus oocyte expressing voltage-dependent potassium channels adhering to a layer of silicon oxide as a simplified model of cell–cell adhesion; here the cell membrane and silicon oxide are separated by a narrow cleft and form a junction of circular shape. We measured directly the extracellular voltage along the diameter of the cleft and investigated its effect on channel gating using a linear array of field effect transistors integrated in the silicon substrate. On this experimental basis we demonstrated that the voltage dependence of potassium channels is strongly affected by adhesion, as can be predicted using a model of a two-dimensional cable and electrodiffusion theory. Computations based on the model showed that along a neuron–astrocyte adhesion contact the opening of voltage-dependent Kv2.1 potassium channels is significantly reduced with respect to identical channels facing an open extracellular space.     

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.The outer hair cell (OHC) is one of two receptor cell types in the organ of Corti, but unlike the inner hair cell it displays electromotile behavior distinct from any other form of cellular motility (1–4). OHC electromotility (eM) arises from the concerted action of millions of molecular motors embedded in the lateral membrane of the cell. They respond directly to membrane voltage and evidence reciprocal activity; namely, they are piezoelectric-like (5–7). Indeed, there is clear evidence that surface area changes accompany state transitions in the motor [see (8)]. The identification of these motors as members of the anion transporter family SLC26 (9), of which prestin is the 5th member (a5), underscores an interesting molecular evolution designed to boost the performance of auditory sensitivity and selectivity. This enhancement is known as cochlear amplification (10).A class of cochlear models requires an electromechanical phase disparity for effective cochlear amplification (11–13), OHC eM lagging receptor potentials. Traditionally, these models assign the mechanism to processes other than the OHC itself. The phase lag provides for the properly timed injection of mechanical force into the cochlear partition to counter viscous detriment. Most molecular models of prestin behavior envision tightly coupled interactions between membrane voltage and eM, arising from sensor charge movements obeying Boltzmann statistics (14–20). Thus, Boltzmann characteristics of sensor charge and eM, namely Q_max /eM_max and Q V_h / eM V_h, are commonly believed to tightly correspond. However, we recently showed significant uncoupling of these characteristics depending on rate and polarity of voltage stimulation and on intracellular chloride level (21). We showed that a slow intermediate transition placed between prestin’s chloride binding transition and the voltage dependent transition responsible for eM could qualitatively account for the data, and we surmised that a molecularly based phase lag should arise. In this study we test this hypothesis by measuring eM at acoustic frequencies and find that indeed substantial frequency dependent phase lags are produced between membrane voltage and eM, showing chloride dependence. An enhanced stretched-exponential kinetic model, termed the meno presto model of prestin, nicely fits the data, whereas a model lacking the intermediate transitions fails.     

Transmitter control of voltage-dependent currents

At one time neurotransmitters were thought of as chemical agents that simply depolarized or hyperpolarized a postsynaptic cell. Now it is known that transmitters can do much more. Biochemical processes, most notably the consequences of activation of adenylate cyclase, are subject to neurotransmitter control. Transmitters can alter a cell's sensitivity to another neurotransmitter; this is exemplified by the action of aspartate in enhancing responses to glutamate. Another action of transmitters is the subject of this review: control of voltage-dependent neuronal currents.Voltage-dependent processes are necessary for the normal function of neuronal systems. Potassium, sodium, and calcium currents that turn on and off with changes in membrane potential are responsible for action potentials and slow-wave (or burst firing) activity. Transmitter control of these ionic currents allows direct synaptic regulation of basic electrophysiological events.Discussion of the voltage-dependent actions of transmitters in neuronal systems will be divided into four areas: (a) broadening and narrowing of action potentials, (b) modulation of burst firing activity, (c) blockade of a voltage-dependent potassium conductance, and (d) induction of a voltage-dependent calcium current. The membrane currents underlying voltage-dependent events will be reviewed only as necessary to understanding transmitter effects. The reader is referred to a recent review for further details on some of these currents (1).     

Developmental Expression of the Outer Hair Cell Motor Prestin in the Mouse

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C _v), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C _v was about 70% of the mature level. C _v saturated at P18 when hearing shows full maturation. On the other hand, C _lin, which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor’s operating voltage midpoint, V _pkcm, tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V _pkcm reached a steady-state level near −67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V _pkcm, may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.     

Voltage-dependent effect of tetraethylammonium on the nicotinic acetylcholine receptors of rat superior cervical ganglion neurons

The effect of tetraethylammonium (TEA) on the currents evoked in neurons of the rat superior cervical ganglion by iontophoretic application of acetylcholine (ACh) was studied using a whole-cell patch-clamp recording technique. Tetraethylammonium was used at a concentration of about 20 µM, providing no blocking effect on the ACh-induced membrane currents at a range of positive membrane potentials and reducing these currents recorded at a range of negative membrane potentials by about half. The blocking effect of TEA increased with hyperpolarization within the –50 to –90 mV membrane potential range, and did not depend on the membrane potential level within a range of 0 to –50 mV. The analysis of dose dependence showed that both the voltage-dependent and the voltage-independent blocking effects are due to TEA competitive action on the ganglionic nicotinic acetylcholine receptors (nAChR). The results suggest that the TEA-induced competitive blockade is voltage-dependent.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 63–66, January–February, 1995.     

Voltage- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

Electromechanical Models of the Outer Hair Cell Composite Membrane

The outer hair cell (OHC) is an extremely specialized cell and its proper functioning is essential for normal mammalian hearing. This article reviews recent developments in theoretical modeling that have increased our knowledge of the operation of this fascinating cell. The earliest models aimed at capturing experimental observations on voltage-induced cellular length changes and capacitance were based on isotropic elasticity and a two-state Boltzmann function. Recent advances in modeling based on the thermodynamics of orthotropic electroelastic materials better capture the cell’s voltage-dependent stiffness, capacitance, interaction with its environment and ability to generate force at high frequencies. While complete models are crucial, simpler continuum models can be derived that retain fidelity over small changes in transmembrane voltage and strains occurring in vivo. By its function in the cochlea, the OHC behaves like a piezoelectric-like actuator, and the main cellular features can be described by piezoelectric models. However, a finer characterization of the cell’s composite wall requires understanding the local mechanical and electrical fields. One of the key questions is the relative contribution of the in-plane and bending modes of electromechanical strains and forces (moments). The latter mode is associated with the flexoelectric effect in curved membranes. New data, including a novel experiment with tethers pulled from the cell membrane, can help in estimating the role of different modes of electromechanical coupling. Despite considerable progress, many problems still confound modelers. Thus, this article will conclude with a discussion of unanswered questions and highlight directions for future research.     

On the frequency response of prestin charge movement in membrane patches

Outer hair cell (OHC) nonlinear membrane capacitance derives from voltage-dependent sensor charge movements within the membrane protein prestin (SLC26a5) that drive OHC electromotility. The ability of the protein to influence hearing depends on its reaction to membrane receptor potentials across auditory frequency. Estimates of prestin’s frequency response have been evaluated by several groups out to tens of kHz in voltage-clamped macro-patches of OHC membrane. The response is a power function of frequency that is down 40 dB at 77 kHz. Despite these observations, concerns remain that the macro-patch approach is flawed due to mechanical constraints of pipette solution column load or patch size itself. In the absence of these influences, prestin’s frequency response is posited by some to be ultrasonic in nature. Here we evaluate the influence of these putative confounding factors on prestin’s frequency response. We show that neither pipette column height nor negative or positive pipette pressure substantially influence total sensor charge frequency response. Additionally, patch surface area has negligible influence. We conclude that the speed of voltage-driven conformational changes in prestin within the plasma membrane is accurately assessed with the macro-patch technique, permitting investigations of membrane characteristics that can substantially alter prestin’s performance bandwidth. We illustrate significant alterations in bandwidth by perturbation of membrane fluidity and chloride anion concentration. Finally, we speculate that OHC membrane characteristics may differ along the tonotopic axis of the cochlea to tune nonlinear membrane capacitance frequency cutoffs.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.     

Non-uniform Distribution of Outer Hair Cell Transmembrane Potential Induced by Extracellular Electric Field

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.