TMC1 Forms the Pore of the Mechanosensitive Transduction Channels in Inner Ear Hair Cells

Abstract

The TMC1 and TMC2 proteins are critical components of the mechanotransduction complex in vertebrate inner-ear hair cells. To understand TMC proteins, we sought insight into the structural properties of TMC1. First, we performed a variety of tests—including size-exclusion chromatography, chemical crosslinking, multi-angle light scattering and cryo electron microscopy— all of which suggest that TMC1 assembles as a dimer. Further, TMC predicted secondary structure and hydrophobicity suggest an architecture with ten transmembrane domains. The dimeric stoichiometry, and topology similarity between TMCs and the TMEM16 family of anion channels and lipid scramblases, suggest that TMCs may have a similar fold. We used I-TASSER to align TMC1 with TMEM16A, for which the atomic structure has been solved. TMEM16A dimerizes at an interface involving the tenth transmembrane domain, and each subunit of the dimer has an separate ion conducting pore, bounded by transmembrane domains S4-S7—a configuration strikingly different from well-known ion channels with a central pore bounded by three to six subunits. To ask whether S4-S7 of TMC1 might also enclose a functional pore, we designed and prepared AAV constructs of TMC1, bearing cysteine mutations, for expression in inner ear hair cells of Tmc1/2-null mice. Through our collaboration with Holt lab, we found that cysteine-modification reagents, when applied acutely, altered mechanosensory currents through these mutant channels in expected ways. Our data together provide evidence that TMC1 forms the pore of sensory transduction channels in auditory hair cells with a permeation pathway lined by residues on S4-S7.