Pioneering work by Holt, Géléoc and Griffith provided strong evidence that the TMC1 and TMC2 proteins are essential constituents of the mechanotransduction complex in vertebrate hair cells. Their precise role, however, remained controversial. In collaboration with the Holt and Sotomayor laboratories, we explored the idea that TMC1 is a pore-forming subunit of this complex.  Homology modeling suggested that the TMC proteins have a structure similar to that of the TMEM16 ion channels and lipid scramblases, which occur as dimers.  We confirmed that TMC1 is a dimer, and—both by analogy to TMEM16A and by molecular dynamics simulations—identified in each subunit a groove composed of transmembrane domains TM4-7 that likely transports cations when the channel is open. We then made cysteine substitutions of key residues in the groove and used AAV vectors to express these mutant TMC1s in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly altered permeation properties of mechanosensory transduction. The data provided compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.

In a new study, we went on to ask whether TMC1 and TMC2 are also the subunits of the complex that sense the stimulus-induced tension in tip links. The AI software AlphaFold2 suggested open and closed structures of TMC1 in which a lateral movement of TM3 and 4 might constitute the gating transition that opens the pore. We made mutations in TM4 and 6, and again used AAV vectors to express mutant channels in hair cells of Tmc1/2-null mice. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining helices TM4 and 6 modified gating—reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel, and so support the idea that TMC1 and TMC2 are the force-sensing subunits of the complex.

Possible gating motion of TMC1. AlphaFold2 predicts slightly different structures for TMC1. Most (lavender) show transmembrane domain 4 (TM4) near TM6, closing off the proposed ion conduction pathway between them. However about a fifth of the AlphaFold2 structures (pink), show TM3 and TM4 positioned further from TM6, creating an open pore. It will be interesting to test this possible gating movement.

PAPERS
Corey DP, Holt JR. (2016) Are TMCs the Mechanotransduction Channels of Vertebrate Hair Cells? J Neurosci. 36:10921-10926.

Pan B, Akyuz N, Liu XP, Asai Y, Nist-Lund C, Kurima K, Derfler BH, György B, Limapichat W, Walujkar S, Wimalasena LN, Sotomayor M, Corey DP, Holt JR. (2018) TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells. Neuron. 99:736-753

Corey DP, Akyuz N, Holt JR. (2019) Function and Dysfunction of TMC Channels in Inner Ear Hair Cells. Cold Spring Harb Perspect Med. 9:a033506.

Akyuz N, Karavitaki KD, Pan B, Tamvakologos PI, Brock KP, Li Y, Marks DS, Corey DP (2021) Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices. bioRxiv 2021.12.30.47456