In VivoStructure–Function Analyses ofCaenorhabditis elegansMEC-4, a Candidate Mechanosensory Ion Channel Subunit

Abstract

Mechanosensory signaling mediated by mechanically gated ion channels constitutes the basis for the senses of touch and hearing and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. We report a genetically based structure-function analysis of the candidate mechanotransducing channel subunit MEC-4, a core component of a touch-sensing complex in Caenorhabditis elegans and a member of the DEG/ENaC superfamily. We identify molecular lesions in 40 EMS-induced mec-4 alleles and further probe residue and domain function using site-directed approaches. Our analysis highlights residues and subdomains critical for MEC-4 activity and suggests possible roles of these in channel assembly and/or function. We describe a class of substitutions that disrupt normal channel activity in touch transduction but remain permissive for neurotoxic channel hyperactivation, and we show that expression of an N-terminal MEC-4 fragment interferes with in vivo channel function. These data advance working models for the MEC-4 mechanotransducing channel and identify residues, unique to MEC-4 or the MEC-4 degenerin subfamily, that might be specifically required for mechanotransducing function. Because many other substitutions identified by our study affect residues conserved within the DEG/ENaC channel superfamily, this work also provides a broad view of structure-function relations in the superfamily as a whole. Because the C. elegans genome encodes representatives of a large number of eukaryotic channel classes, we suggest that similar genetic-based structure-activity studies might be generally applied to generate insight into the in vivo function of diverse channel types.

Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

“…For example, mutations within the finger domain of DEG-1 and UNC-8 induced channel superactivation (35,36). In MEC-4, mutations within the finger domain lead to a disruption of mechanosensation ability in Caenorhabditis elegans (37). In ASIC channels, the lower finger domain associated with the thumb and ␤-ball forms an acidic pocket, acting as one of the proton sensors (10).…”

Touch sensitivity in Caenorhabditis elegans

“…2) [44]. The second transmembrane domain (TMII) and the sequence preceding it are thought to contribute to the pore of the channel; mutations in these regions cause constitutive channel activity, blockage of the constitutive activity, or changes in ion selectivity [36,37,57]. The mutations that cause constitutive channel activity were first identified as changes in MEC-4, MEC-10, and other C. elegans degenerins that cause neurodegeneration [16,21,38].…”

“…The most C-terminal CRD, which is highly conserved, exhibits similarity to venom neurotoxin, suggesting a possible role in modulating DEG/ENaC channel gating [83]. mec-4 mutations in this region disrupt channel function and may affect appropriate gating [37]. Finally, parts of the cytoplasmic COOH-terminal domain of MEC-4 may be required for channel trafficking or maintenance of the channel in the cell surface [67].…”

Pickpocket1 Is an Ionotropic Molecular Sensory Transducer

“…Although there are many examples of ion channel receptors that can respond to sensory stimuli (1)(2)(3)(4)(5)(6)(7)(8), there are few examples where activation of these channels can drive neuronal activity.…”