<b>Supporting data for "</b><b>Auxiliary Subunits Orchestrate the Regulation, Expression, and Function of TMC Channels"</b>

<p dir="ltr">The functional identity of ion channels is primarily determined by the properties conferred by their pore-forming subunits. However, the functional diversity of certain pleiotropic proteins that influence multiple phenotypic traits is often granted by their combination with non-pore-forming auxiliary subunits [1]. Additionally, auxiliary subunits play an essential role in fine-tuning the gating, trafficking, localization, and stability of channels. In this thesis, we provide evidence that auxiliary subunits are critical in regulating TMC channels, the molecular receptor of hearing in mammals.</p><p><br></p><p dir="ltr">By using the <i>Caenorhabditis elegans</i><i> </i>as a model animal, we studied the assembly of TMC complexes, including pore-forming subunits TMC-1/2 and auxiliary subunits CALM-1, TMIE, and a newly discovered arrestin protein ARRD-6 [2]. We found that the genetic removal of auxiliary subunits would disrupt functions mediated by TMC complexes (leaky channel in mediating membrane potential, mechanosensation in nose touch reported by others), while the alkali-sensing ability of TMC-1 is preserved. Our data challenge the published result that TMC-1 is a high-salt sensor, indicating that TMC-1 is also a bona fide alkaline receptor.</p><p><br></p><p dir="ltr">Our study elucidates the biological function of ARRD-6 as a downregulator of the TMC complex, finding that the knockout of ARRD-6 rescued the deficit of <i>tmc</i> mutants, increased the abundance of TMC-1/2, and slowed the adaptation of TMC-1-mediated alkali-sensing, but did not cause any loss-of-function phenotype of <i>tmc</i>. Overexpression of the ARRD-6 by using its own genomic DNA or tissue-specific promoter phenocopied the <i>tmc</i> knockout phenotype. Mechanistic investigations indicated that ARRD-6 and its murine homolog likely function analogously to canonical beta-arrestins by engaging with ubiquitination and endocytosis machinery. Knockout of the mouse arrestin protein ARRDC4 resulted in significant hearing loss compared to wild-type mice after noise exposure, revealing a protective role of arrestin in hearing and paralleling the established role of visual arrestin in phototransduction. Collectively, we identified an evolutionarily conserved regulator of TMC proteins, influencing TMC protein function across sensory modalities.</p><p><br></p><p dir="ltr">In contrast, knockout of <i>tmie</i><i> </i>and <i>calm-1</i><i> </i>evoked profound deficits in TMC-1/2-mediated egg-laying and nose touch behavior [3], affected the trafficking, expression pattern, and stabilization of TMC-1/2, and fastened the adaptation to repetitive alkaline stimuli through unknown mechanisms. Our results and published literature support the idea that TMIE and CALM-1/CIB2 are functional auxiliary subunits involved in the transduction of the TMC complex.</p><p><br></p><p dir="ltr">As side projects, we demonstrated that heterologous expression of truncated human TMC1 could be localized to the plasma membrane in <i>C. elegans,</i><i> </i>making it an invaluable tool for functional characterization of TMC proteins. Inducible expression of both CeTMC-1 and HsTMC1 was restricted in TMC-1-negative cells, revealing a unique negative regulatory system for TMC proteins.</p><p><br></p><p dir="ltr">In summary, my doctoral research established a regulatory network of the mammalian hearing receptor TMC proteins, uncovered a novel role of arrestins in regulating the mechanogated channels, and systematically characterized the role of components in the worm TMC complex. By integrating the advantages of two well-defined model organisms, my work sheds light on the poorly understood regulation of the mammalian mechanosensor and potentiates prevention or therapy for noise-induced hearing loss.</p>