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The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.