In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit

Mechanosensory signaling mediated by mechanically gated ion channels consti tutes the basis for the senses of touch and hearing and contributes fundame ntally to the development and homeostasis of all organisms. Despite this pr ofound importance in biology, little is known of the molecular identities o r functional requirements of mechanically gated ion channels. We report a g enetically based structure-function analysis of the candidate mechanotransd ucing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. We identi fy molecular lesions in 40 EMS-induced mec-4 alleles and further probe resi due and domain function using site-directed approaches. Our analysis highli ghts residues and subdomains critical for MEC-4 activity and suggests possi ble roles of these in channel assembly and/or function. We describe a class of substitutions that disrupt normal channel activity in touch transductio n but remain permissive for neurotoxic channel hyperactivation, and we show that expression of an N-terminal MEC-4 fragment interferes with in vivo ch annel function. These data advance working models for the MEC-4 mechanotran sducing channel and identify residues, unique to MEC-4 or the MEC-4 degener in subfamily, that might be specifically required for mechanotransducing fu nction. Because many other substitutions identified by our study affect res idues conserved within the DEG/ENaC channel superfamily, this work also pro vides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large num ber of eukaryotic channel classes, we suggest that similar genetic-based st ructure-activity studies might be generally applied to generate insight int o the in vivo function of diverse channel types.