MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: auxiliary subunits that enable channel activity.
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.
Brown
Austin L
AL
Biophysics Program, Stanford University, Stanford, CA 94305, USA.
Liao
Zhiwen
Z
Goodman
Miriam B
MB
D000818:Animals; D043303:Aryldialkylphosphatase; D017173:Caenorhabditis elegans / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D002918:Chymotrypsin / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D054878:Lipoylation / Q000502:physiology; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015347:RNA Probes / Q000032:analysis; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis
D029742:Caenorhabditis elegans Proteins; D053503:Epithelial Sodium Channels; C096705:MEC-2 protein, C elegans; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D015347:RNA Probes; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D002784:Cholesterol; C470181:MEC-6 protein, C elegans; D043303:Aryldialkylphosphatase; D002918:Chymotrypsin; D002118:Calcium