Abstract
The Caenorhabditis elegans MEC-4/MEC-10 channel mediates the worm's response to gentle body touch and is activated by laminar shear stress (LSS) when expressed in Xenopus oocytes. Substitutions at multiple sites within the second transmembrane domain (TM2) of MEC-4 or MEC-10 abolish the gentle touch response in worms, but the roles of these residues in mechanosensing are unclear. The present study therefore examined the role of specific MEC-4 and MEC-10 TM2 residues in the channel's response to LSS. We found that introducing mutations within the TM2 of MEC-4 or MEC-10 not only altered channel activity, but also affected the channel's response to LSS. This response was enhanced by Cys substitutions at selected MEC-4 sites (Phe715, Gly716, Gln718, and Leu719) between the degenerin and the putative amiloride-binding sites in this subunit. In contrast, the LSS response was largely blunted in MEC-10 variants bearing single Cys substitutions in the regions preceding and following the amiloride-binding site (Gly677-Leu681), as well as with four MEC-10 touch-deficient mutations that introduced charged residues into the TM2 domain. An enhanced response to LSS was observed with a MEC-10 mutation in the putative selectivity filter. Overall, MEC-4 or MEC-10 mutants that altered the channel's LSS response are primarily clustered between the degenerin site and the selectivity filter, a region that probably forms the narrowest portion of the channel pore. Our results suggest that pore-lining residues of MEC-4 and MEC-10 have important yet different roles in tuning the channel's response to mechanical forces.
Highlights
The Caenorhabditis elegans MEC-4/MEC-10 channel mediates the worm’s response to gentle body touch and is activated by laminar shear stress (LSS) when expressed in Xenopus oocytes
C. elegans MEC-4/MEC-10 channels are activated by flowmediated LSS in Xenopus oocytes, and MEC-10 is required for a robust LSS response [14]
We examined the role of TM2 residues of MEC-4 and MEC-10 in the channel’s LSS response
Summary
At the core of this mechanotransduction apparatus is a mechanically gated ion channel formed by MEC-4 and MEC-10 subunits, members of the ENaC/degenerin family [8, 9]. A large number of touch-deficient alleles have mutations within the pore-lining region of MEC-4 (16 alleles at 13 sites) and MEC-10 (five alleles), highlighting the importance of the channel’s gate and pore in sensing mechanical stimuli [23]. Pore-lining residues within the second transmembrane domain (TM2) of ENaC subunits are important for modulating channel gating in response to flow-mediated laminar shear stress (LSS) (24 – 26). The present study used the LSS response to examine the role of TM2 domains of the two pore-forming subunits, MEC-4 and MEC-10, in channel gating by a mechanical force. Our data support the hypothesis that pore-lining residues within the TM2 domain of MEC-4 or MEC-10 have important yet different roles in accommodating conformational rearrangements within the channel pore during channel gating in response to mechanical forces
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