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

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, Qmax . Prestin-transfected cells also showed reversible reduction in Qmax . For OHCs, intracellular 9-AC application as well as reduced intracellular pH had no detectable effect on the reduction in Qmax 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 Qmax . 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.