Subunit determination of the conductance of hair-cell mechanotransducer channels

Abstract

Cochlear hair cells convert sound stimuli into electrical signals by gating of mechanically sensitive ion channels in their stereociliary (hair) bundle. The molecular identity of this ion channel is still unclear, but its properties are modulated by accessory proteins. Two such proteins are transmembrane channel-like protein isoform 1 (TMC1) and tetraspan membrane protein of hair cell stereocilia (TMHS, also known as lipoma HMGIC fusion partner-like 5, LHFPL5), both thought to be integral components of the mechanotransduction machinery. Here we show that, in mice harboring an Lhfpl5 null mutation, the unitary conductance of outer hair cell mechanotransducer (MT) channels was reduced relative to wild type, and the tonotopic gradient in conductance, where channels from the cochlear base are nearly twice as conducting as those at the apex, was almost absent. The macroscopic MT current in these mutants was attenuated and the tonotopic gradient in amplitude was also lost, although the current was not completely extinguished. The consequences of Lhfpl5 mutation mirror those due to Tmc1 mutation, suggesting a part of the MT-channel conferring a large and tonotopically variable conductance is similarly disrupted in the absence of Lhfpl5 or Tmc1. Immunolabelling demonstrated TMC1 throughout the stereociliary bundles in wild type but not in Lhfpl5 mutants, implying the channel effect of Lhfpl5 mutations stems from down-regulation of TMC1. Both LHFPL5 and TMC1 were shown to interact with protocadherin-15, a component of the tip link, which applies force to the MT channel. We propose that titration of the TMC1 content of the MT channel sets the gradient in unitary conductance along the cochlea.cochlea | mechanotransducer channels | TMC1 | hair cell | LHFPL5 C ochlear hair cells detect sound stimuli by submicron vibrations of their stereociliary (hair) bundles. The stereocilia are arranged in three to four rows, stepped in height and interconnected by extracellular linkages; the most important for transduction are the tip links (1, 2), composed of cadherin-23 and protocadherin-15 (3, 4). During bundle displacements, they transmit force to activate mechanotransducer (MT) ion channels near the insertion of protocadherin-15 at the lower end of the tip link into the stereociliary tip (5, 6). The molecular identity of the pore-forming subunit of the ion channel is still controversial, but there has been a recent proposal that transmembrane channellike protein isoforms 1 and 2 (TMC1 and TMC2) (7, 8) are possible candidates (9, 10); mutations of these proteins can alter the Ca 2+ selectivity and single-channel conductance of the MT channels, implying that TMC proteins can influence ion conduction through the pore (10-12). However, in Tmc1/Tmc2 double mutants, large mechanically sensitive currents can still be evoked and flow through channels similar to native MT channels (13). Thus, an alternate view is that the TMC1 and TMC2 are accessory but not pore-forming subunits of the channel.Another likely component of the transduction mac...

Development and localization of reverse-polarity mechanotransducer channels in cochlear hair cells

“…In apical OHCs, both are cation channels that pass Ca 2+ with P Ca /P Cs = ∼5.8 in normal and ∼1.5 for reverse-polarity channels; both are blocked by external FM1-43 with IC 50 of ∼3 μM in normal and 26 μM in unconventional channels; the unitary conductances are similar in apical OHCs, with values in 1.5 mM Ca 2+ of 63 pS in normal and 61 pS for unconventional channels. However, in basal OHCs the normal channels are about twofold larger than the reverse-polarity channels, a difference that depends on the presence of TMC1, which may contribute to an external vestibule (9,31). In both cases the channels are blocked by extracellular Ca 2+ , lowering its concentration to a few tens of micromolars, increasing channel conductance by 50%.…”

Cadherins in the Auditory Sensory Organ

“…Yeast two-hybrid and coimmunoprecipitation experiments have shown that the CD1 and CD3 isoforms of protocadherin-15 interact directly with Tmc1 and Tmie (Maeda et al 2014). All three isoforms of protocadherin-15 were found to coimmunoprecipitate with both TMC1 and TMC2 (Beurg et al 2015). In another study, Zhao and colleagues showed that the CD2 isoform bound Tmie directly, but that the CD1 and CD3 isoforms bound Tmie only indirectly, via Tmhs see Fig.…”

The tip-link molecular complex of the auditory mechano-electrical transduction machinery

“…These four proteins are Tmc1 (transmembrane channel protein 1), Tmc2 (transmembrane channel protein 2) (Beurg et al, 2014(Beurg et al, , 2015Kawashima et al, 2011;Pan et al, 2013), Tmhs (tetraspan membrane protein of hair cell stereocilia, also know as Lhfpl5) (Beurg et al, 2015;Longo-Guess et al, 2007, 2005Xiong et al, 2012) and Tmie (transmembrane protein of inner ear hair cells) (Karuppasamy et al, 2012;Mitchem et al, 2002;Park et al, 2013; Su CD3-specific sequence (encoded by exon 39) is indicated in yellow, and the sequence encoded by exon 37 is indicated in green. Protein sequences encoded by phases other than the conventional phase are indicated in gray.…”