Chloride ions have been hypothesized to interact with the membrane outer hair cell (OHC) motor protein, prestin on its intracellular domain to confer voltage sensitivity (Oliver et al., 2001). Thus, we hypothesized previously that transmembrane chloride movements via the lateral membrane conductance of the cell,G_metL, could serve to underlie cochlear amplification in the mammal. Here, we report on experimental manipulations of chloride-dependent OHC motor activityin vitroandin vivo.In vitro, we focused on the signature electrical characteristic of the motor, the nonlinear capacitance of the cell. Using the well known ototoxicant, salicylate, which competes with the putative anion binding or interaction site of prestin to assess level-dependent interactions of chloride with prestin, we determined that the resting level of chloride in OHCs is near or below 10 mm, whereas perilymphatic levels are known to be ∼140 mm. With this observation, we sought to determine the effects of perilymphatic chloride level manipulations of basilar membrane amplification in the living guinea pig. By either direct basolateral perfusion of the OHC with altered chloride content perilymphatic solutions or by the use of tributyltin, a chloride ionophore, we found alterations in OHC electromechanical activity and cochlear amplification, which are fully reversible. Because these anionic manipulations do not impact on the cation selective stereociliary process or the endolymphatic potential, our data lend additional support to the argument that prestin activity dominates the process of mammalian cochlear amplification.

Isolated outer hair cells (OHCs) from the guinea‐pig were whole‐cell voltage clamped to study the influence of initial voltage on the voltage dependence of motility‐related gating current or, equivalently, on the voltage dependence of membrane capacitance. Prepulse delivery caused changes in the magnitude of motility‐related gating currents, which are due predominantly to shifts in the voltage at peak capacitance (V_pkCm). Depolarization shifts V_pkCm in the hyperpolarizing direction, and hyperpolarization does the opposite. The mean shift between ‐120 and +40 mV prepulse states with long‐term holding potentials (> 2 min) at −80 mV was 14.67 ± 0.95 mV (n= 10; mean ± s.e.m.). The effect of initial membrane potential is sigmoidal, with a voltage dependence of 23 mV per e‐fold change in V_pkCm, and maximum slope within the physiological range of OHC resting potentials. This indicates that the cell is poised to respond maximally to changes in resting potential. The kinetics of prepulse effects are slow compared with motility‐related gating current kinetics. High‐resolution measurement of membrane capacitance (C_m) using two voltage sinusoids indicates that shifts in V_pkCm induce C_m changes with time courses fitted by two exponentials (τ₀, 0.070 ± 0.003 s; τ₁, 1.28 ± 0.07 s; A₀, 1.54 ± 0.13 pF; A₁, 1.51 ± 0.13 pF; means ± s.e.m.; n= 22; step from +50 to −80 mV). Recovery of prepulse effects exhibits a similar time course. Prepulse effects are resistant to intracellular enzymatic digestion, to fast intracellular calcium buffers, and to intracellular pressure. Through modelling, we indicate how the effect may be explained by an intrinsic voltage‐induced tension generated by the molecular motors residing in the lateral membrane.

Whole cell voltage clamp and freeze fracture were used to study the electrophysiological and ultrastructural correlates of the outer hair cell (OHC) lateral membrane molecular motors. We find that specific voltage-dependent capacitance, which derives from motility-related charge movement, increases as cell length decreases. This increasing non-linear charge density predicts a corresponding increase in sensor-motor density. However, while OHC lateral membrane particle density increases, a quantitative correspondence is absent. Thus, the presumed equivalence of particle and motor is questionable. The data more importantly indicate that whereas the voltage driving OHC motility, i.e. the receptor potential, may decrease with frequency due to the OHC's low-pass membrane filter, the electrical energy (Q x V) supplied to the lateral membrane will tend to remain stable. This conservation of energy delivery is likely crucial for the function of the cochlear amplifier at high frequencies.