The conductance of TMC1-containing mechanotransducer channels and the roles of LHFPL5 and TMIE in cochlear hair cells.

Abstract

Transmembrane channel-like protein 1 (TMC1) is thought to form the ion-conducting pore of the mechanotransducer (MET) channel in auditory hair cells, assisted by accessory subunits TMIE, LHFPL5 and CIB2. LHFPL5 is most likely connected to the carboxy terminus of PCDH15 at the lower end of the tip link. The organization of the channel complex is still unclear, but a TMC1 structure based on homology with TMEM16A indicates ten transmembrane (TM) domains with the pore lined by TMs 4-7. Using single channel analysis and ionic permeability measurements, we characterized six missense mutations in the purported pore region of mouse TMC1. MET currents could be recorded in cochlear hair cells of neonatal mutant mice, showing all mutations reduced the Ca2+ permeability of the channel. In addition, Tmc1 p.E520Q and Tmc1 p.D528N reduced channel conductance whereas Tmc1 p.W554L and Tmc1 p.D569N lowered channel expression without affecting the conductance. The consequences of these mutations endorse TMC1 as the pore of the MET channel, the mutations occurring in transmembrane domains 6-7. The accessory subunits LHFPL5 and TMIE are thought to be involved in targeting TMC1 to the tips of the stereocilia. We found sufficient expression of TMC1 in hair cells of Lhfpl5 and Tmie knockout mice to characterize the remaining MET channels, which could still be gated by hair bundle deflections. Single-channel conductance was unaffected in Lhfpl5-/- but was reduced in Tmie‑/-, implying TMIE very likely contributes to the pore. Both the working range and half-saturation point of the residual MET current in Lhfpl5-/- were substantially increased, suggesting that LHFPL5 is part of the mechanical coupling between the tip-link and the MET channel.