Initial Functional and Structural Analysis of Plant Mechanosensitive Channel MSL10

Abstract

MSL10 is the first mechanosensitive channel from Arabidopsis thaliana to be electrophysiologically characterized. MSL10 is a member of the MscS family of which there are 10 in Arabidopsis. Though MSL10 is topologically different from known bacterial MS channels, its most C-terminal TM helix shows moderate homology with E. coli MscS's pore-lining domain. MSL10 mechanosensitivity has been shown both in exogenous (Xenopus oocytes) and endogenous systems. However, nothing was known about the structure and organization of any eukaryotic MscS homolog. Using mutational analysis, electrophysiology, and in planta assays, we identify residues important for MSL10 function, concentrating on its cytoplasmic N-terminus, 6 cysteine residues located both on cytoplasmic and extracellular sides and in TM helices, and the putative pore-lining domain. Recently we have identified MSL10's N-terminus as a multiple phosphorylation site, which suggests its importance for channel regulation. A set of mutants mimicking either phosphorylated or non-phosphorylated forms of the MSL10 protein was tested by patch-clamp when expressed in Xenopus oocytes and simultaneously studied in planta. According to our model, phosphorylation of the N-terminus alone does not affect channel function. Instead, it mediates an unknown step in cell death signaling. MD simulations have suggested an impact of phosphorylation on the structure of MSL10's cytoplasmic N-terminus. We have also demonstrated that cysteine pairs on the cytoplasmic (C205, C283) and on the extracellular (C95, C581) side of the channel do not form bonds critical for channel assembly and gating. And finally, mutations introduced into MSL10's putative pore-lining domain (V549S, F553S; F553L and F563L) allowed us to identify residues that affect channel gating and stability. Therefore, this work has revealed several important structure-function relations for the newly characterized family of MS channels in plants.