Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells.

UNLABELLED The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1(Bth/Bth)) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca(2+) permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1(Bth/Bth) OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca(2+) dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1(Bth/Bth) OHCs, whereas raising the intracellular concentration of the Ca(2+) chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca(2+) permeability of the mutated MET channel indirectly causing the Ca(2+) sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca(2+) influx as a compensatory mechanism. SIGNIFICANCE STATEMENT In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca(2+) permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca(2+)-dependent adaptation appear stronger, in compensation for the reduced Ca(2+) entry.