The chloride-channel blocker 9-anthracenecarboxylic acid reduces the nonlinear capacitance of prestin-associated charge movement.

Abstract

The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride‐sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9‐anthracenecarboxylic acid (9‐AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9‐AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea‐pig cochlea and of prestin‐transfected human embryonic kidney 293 (HEK 293) cells. Extracellular application of 9‐AC caused reversible, dose‐dependent and chloride‐sensitive reduction in OHC nonlinear charge transfer, Q max. Prestin‐transfected cells also showed reversible reduction in Q max. For OHCs, intracellular 9‐AC application as well as reduced intracellular pH had no detectable effect on the reduction in Q max by extracellularly applied 9‐AC. In the prestin‐transfected cells, cytosolic application of 9‐AC approximately halved the blocking efficacy of extracellularly applied 9‐AC. OHC inside‐out patches presented the whole‐cell blocking characteristics. Disruption of the cytoskeleton by preventing actin polymerization with latrunculin A or by decoupling of spectrin from actin with diamide did not affect the 9‐AC‐evoked reduction in Q max. We conclude that 9‐AC acts on the electromechanical transducer principally by interaction with prestin rather than acting via the cytoskeleton, chloride channels or pH. The 9‐AC block presents characteristics in common with salicylate, but is almost an order of magnitude faster. 9‐AC provides a new tool for elucidating the molecular dynamics of prestin function.