DegFlow FINAL 2

Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10 and accessory proteins. To define the role of these subunits in the channel’s response to mechanical force, we expressed degenerin channels comprised of MEC-4 and MEC-10 in Xenopus oocytes and examined its response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C. elegans degenerin channels are sensitive to LSS. MEC-10 is required for a robust LSS response as the response was largely blunted in oocytes expressing homomeric MEC 4 or MEC-4d channels. We examined a series of MEC-10/MEC-4 chimeras to identify specific domains (amino terminus, first transmembrane domain and extracellular domain) and sites (residues 130-132 and 134-137) within MEC-10 that are required for a robust response to shear stress. In addition, the LSS response was largely abolished by MEC-10 mutations encoded by a touch-insensitive mec-10 allele, providing a correlation between the channel’s responses to two different mechanical forces. Our findings suggest that MEC-10 has an important role in the channel’s response to mechanical forces. The worm Caenorhabditis elegans (C. elegans) senses a variety of mechanical stimuli, including gentle body touch, harsh body touch, nose touch and texture (1,2). Gentle touch is sensed via a multi-protein mechanosensitive channel complex expressed in six touch receptor neurons (TRNs) along the worm body (2,3). Worms bearing mutations in genes encoding the channel complex exhibit impaired mechanosensation (4,5). At the core of this mechanosensitive channel complex are the two pore-forming subunits, MEC-4 and MEC-10 (4,6). MEC-4 can form either a homomeric channel by itself or heterotrimeric channels with MEC-10, comprised of two MEC-4 subunits and a MEC-10 subunit (7). Together, they form a channel that is permeable to Na and Ca (8,9), and sensitive to amiloride and specific amiloride derivatives (10). MEC-4 and MEC-10 are members of the epithelial sodium channel (ENaC)/degenerin family, which encodes a group of structurally related cation channels that are involved in a variety of fundamental cellular processes (11,12). Members of this family include (i) C. elegans degenerins (4,6,13); (ii) Drosophila pickpockets (14-16); and (iii) mammalian ENaCs and acid sensing ion channels (ASICs) (17-20). These channels sense specific factors in the external environment that modulate their channel activity (21,22). It has been proposed that C. elegans mechanosensitive channels are quiescent at rest. Upon mechanical stimulation, channels open transiently to http://www.jbc.org/cgi/doi/10.1074/jbc.M116.718031 The latest version is at JBC Papers in Press. Published on May 4, 2016 as Manuscript M116.718031 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 6, 2017 hp://w w w .jb.org/ D ow nladed from