Search

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex., Competing Interests: Competing Interest Statement The authors declare no competing interests.

Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily-conserved family of ion channels whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila . The molecular features that tune homologous TMC ion channel complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here we present the single particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans . The complex is composed of two copies each of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex reveals differences in subunit composition and highlights conserved protein-lipid interactions, as well as other structural features, that together suggest a mechanism for TMC-mediated mechanosensory transduction., Competing Interests: Competing Interest Statement: The authors declare no competing interests.