Localization of Anomalous Mechano-Sensitive Ion Channels in Cochlear Hair Cells

Abstract

Two types of mechanosensitive (MS) currents have been described in cochlear hair cells: conventional MS currents evoked by displacements of the hair bundle towards its tallest edge, and anomalous MS currents, elicited by bundle displacements in the opposite direction, referred to as reversed-polarity currents. Conventional MS transducer channels are located at the bottom end of the tip links, but reverse-polarity MS currents persist after severing tip links with BAPTA, suggesting a different site for this channel. We investigated localization of reverse-polarity MS channels using cell-attached patch recordings on cochlear outer hair cells (OHC) of neonatal mice, and also by detecting calcium influx with Fluo5F indicator using swept-field confocal imaging. Single mechano-sensitive channels were recorded in cell-attached mode on the apical surface around the base of the hair bundle and were activated by suction through the patch pipette. In OHCs of wild-type mice, MS channels appeared a few minutes after BAPTA exposure with a mean conductance of 61 ± 6 pS in Na-saline with 1.5 mM CaCl2 and 94 ± 13 pS in Na-saline with reduced, 0.07 mM, CaCl2. Their reversal potential was 0 mV as expected for a non-selective cationic channel. Similar MS channels were also seen in Tmc1:Tmc2 double mutants, with mean conductance of 58 ± 4 pS in 1.5 mM CaCl2. In both cases, the apical surface of the OHC was confirmed as the main site of calcium entry during reverse polarity MS currents by imaging calcium influx. The properties of the reverse-polarity MS channels closely resemble conventional MS channels but they are concentrated on the apical plasma membrane of the hair cell. We suggest they reflect insertion of new MS channels before their transport up the bundle to the transduction complex.