Gain-of-Function Mutations in the MEC-4 DEG/ENaC Sensory Mechanotransduction Channel Alter Gating and Drug Blockade

Abstract

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.

Intersubunit interactions between mutant DEG/ENaCs induce synthetic neurotoxicity

“…Consistent with the previously reported current-suppressing effect of MEC-10( þ ) on MEC-4(d) currents, 11,13,20 we find that the coexpression of MEC-4(A149V) with MEC-10( þ ) is associated with currents that are smaller relative to MEC-4(A149V) alone. If the native MEC-4(A149V) þ MEC-10( þ ) channel has altered electrophysiological properties in vivo, changes are not substantial enough to disrupt the function in touch sensation, as the mec-4(bz301) mutant is touch sensitive and mec-4 function is required for this behavior (Figure 4b).…”

“…has been electrophysiologically characterized in Xenopus oocytes 10,11,13,20 (see also Figure 5a). In brief, this work showed that (1) channels including the MEC-4(d) subunit conduct markedly elevated Na þ and Ca 2 þ currents relative to MEC-4( þ ) channels (note that MEC-4( þ ) conducts only barely detectable currents); (2) MEC-10( þ ) and MEC-10(d) do not conduct current as homomeric channels; (3) MEC-10 is not essential for MEC-4(d) channel conductance and dampens MEC channel currents when present; and (4) stomatin MEC-2 and paraoxonaserelated MEC-6 subunits increase current.…”

Activation of the Caenorhabditis elegans Degenerin Channel by Shear Stress Requires the MEC-10 Subunit

“…MEC-4 can form either a homomeric channel by itself or heterotrimeric channels with MEC-10, comprising two MEC-4 subunits and a MEC-10 subunit (7). Together, they form a channel that is permeable to Na ϩ and Ca 2ϩ (8,9) and sensitive to amiloride and specific amiloride derivatives (10).…”

Subunit composition of a DEG/ENaC mechanosensory channel of Caenorhabditis elegans

“…Second, MEC-2 and MEC-6 increased the activity of MEC-4(d) channels 5 d after injection of their cRNAs into Xenopus oocytes without apparently changing the amount of surface-localized MEC-4(d) protein (8)(9)(10)(11). [The dominant mec-4(d) mutation, which results in an A713T substitution, produces a hyperactive channel that is constitutively active (9,12,13) and leads to TRN degeneration.] Third, MEC-2 and MEC-6 coimmunoprecipitated with MEC-4(d), MEC-10, and each other in heterologous cells (8)(9)(10).…”