Control of Mammalian Cochlear Amplification by Chloride Anions

Abstract

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 activity in vitro and in 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 ϳ140 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.

Disparities in voltage-sensor charge and electromotility imply slow chloride-driven state transitions in the solute carrier SLC26a5

“…Chloride levels were set to bracket the intracellular level in intact OHCs, namely ∼10 mM (6). An ionic blocking solution was used to remove ionic currents, allowing valid measures of membrane capacitance.…”

“…Following the molecular identification of prestin (3), an SLC26 family member (a5) that recapitulates the electromechanical properties of the native OHC's voltage sensor/motor (4,5), a preponderance of evidence pointed to prestin-based electromotility (eM) as the basis of this boost (6,7). During the last couple of decades, measures of nonlinear charge movement or capacitance (NLC), an electrical correlate of voltage-dependent conformational changes within the motor protein prestin, have served as surrogate, indeed as signature, of voltage-evoked eM of the OHC (8)(9)(10)(11)(12).…”

“…That is, a dissociation in magnitude and voltage dependence of NLC and eM is revealed by lowering intracellular chloride to physiological levels (≤ 10 mM; ref. 6), all previous comparisons having been made with abnormally high intracellular chloride levels. Furthermore, this chloride effect, which presents as a function of rate and polarity of voltage stimulation, uncovers a mechanism predicted to be frequency dependent in vivo, and likely plays an important role in frequency selectivity and efficiency of cochlear amplification.…”

Dissection of the Mechanical Impedance Components of the Outer Hair Cell Using a Chloride-Channel Blocker

“…In the presence of 135-mM intracellular chloride, the dose-dependence of the extracellularly applied 9AC on OHCs and on HEK293 cells is similar to that for salicylate [12] suggesting similar blocking mechanisms.…”

“…Therefore, 9AC is probably not competing for the same binding site as chloride ions. However, the extracellular and intracellular chloride concentrations were not changed in parallel, as in other OHC chloride experiments [12]. Having only changed the extracellular concentration, we cannot exclude the possibility that chloride and 9AC bind to the same site.…”