title: 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.
authors.affiliation: Biophysics Program, Stanford University School of Medicine, CA 94305, USA.
authors.forename: Austin L
authors.initials: AL
authors.lastname: Brown
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Fernandez-Illescas
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Liao
authors.forename: Miriam B
authors.initials: MB
authors.lastname: Goodman
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D021122:Protein Subunits; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D029742:Caenorhabditis elegans Proteins; C094927:Deg-1 protein, C elegans; D053503:Epithelial Sodium Channels; C115871:Mec-4 protein, C elegans; D008565:Membrane Proteins; D021122:Protein Subunits; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C013407:benzamil; C085942:MEC-10 protein, C elegans; D000584:Amiloride