Reconstitution of an ion channel complex needed for mechanosensation in Caenorhabditis elegans

The molecules responsible for sensory mechanotransduction—the process that underlies touch, hearing, balance, and proprioreception—in animals are poorly understood. Genetic studies in Caenorhabditis elegans have identified twelve mec genes needed for the function of six touch receptor cells. Many of these genes interact genetically, and most are co-expressed in the same receptors. Genetic characterization of mec-4 and mec-10, and sequence similarity to amiloride-sensitive epithelial sodium channels, suggested that they express ion channels at the core of a mechanotransduction complex. Genetic studies suggested the MEC-6 protein is necessary for MEC-4 and MEC-10 activity, and the stomatin-like protein MEC-2 modulates these presumptive channel components.; I tested the hypothesis that MEC-4 and MEC-10 are ion channels and the hypothesis that MEC-2 and MEC-6 functionally interact with these channels. I analyzed the heterologous expression of these four proteins by using the two-electrode voltage clamp to assay amiloride-sensitive current in Xenopus oocytes. I have found that oocytes express amiloride-sensitive current when they are co-injected with RNAs that encode MEC-4 and MEC-10 proteins that have gain-of-function mutations that cause touch cell degeneration. The degeneration-inducing form (‘d’ form) of MEC-4 can also produce detectable amiloride-sensitive currents alone. The inclusion of MEC-2, MEC-6 or both enhances the current amplitude produced by MEC-4d or MEC-4d+MEC-10d. The enhancement by the co-expression of MEC-2 and MEC-6 is synergistic. Protein surface expression assays revealed that an increase in channel protein surface expression could not account for the increase in channel activity. Amiloride-dose response analyses found MEC-10d interacts with MEC-4d to increase amiloride-sensitivity by about eight-fold. The dose-response data suggest that co-expression of all four proteins are present produce a single amiloride binding site, which suggests all four proteins can co-assemble. Co-immunoprecipitation studies form heterologous cells biochemically confirm that these proteins are closely associated. These data support the conclusion that MEC-4, MEC-10, MEC-2, and MEC-6 can form an ion channel complex and suggest that MEC-2 and MEC-6 may directly regulate ion channel gating and/or conduction. The reconstitution of MEC-protein channel activity in Xenopus oocytes is an important step toward understanding how these proteins contribute to mechanotransduction.