Nonpolar residues in the presumptive pore-lining helix of mechanosensitive channel MSL10 influence channel behavior and establish a nonconducting function.

Grigory Maksaev,Simran Ohri,Jennette M Shoots,Elizabeth S Haswell

doi:10.1002/pld3.59

Abstract

Mechanosensitive (MS) ion channels provide a universal mechanism for sensing and responding to increased membrane tension. MscS‐like (MSL) 10 is a relatively well‐studied MS ion channel from Arabidopsis thaliana that is implicated in cell death signaling. The relationship between the amino acid sequence of MSL10 and its conductance, gating tension, and opening and closing kinetics remains unstudied. Here, we identify several nonpolar residues in the presumptive pore‐lining transmembrane helix of MSL10 (TM6) that contribute to these basic channel properties. F553 and I554 are essential for wild type channel conductance and the stability of the open state. G556, a glycine residue located at a predicted kink in TM6, is essential for channel conductance. The increased tension sensitivity of MSL10 compared to close homolog MSL8 may be attributed to F563, but other channel characteristics appear to be dictated by more global differences in structure. Finally, MSL10 F553V and MSL10 G556V provided the necessary tools to establish that MSL10's ability to trigger cell death is independent of its ion channel function.

Highlights

The ability to respond to mechanical stimuli is an ancient and intrinsic property of cells (Anishkin, Loukin, Teng, & Kung, 2014; Booth, Miller, Mu€ller, & Lehtovirta-Morley, 2015)
We introduced F544V, F553W, F553L, F553V, and F563L mutations into the MSL10 coding sequence of pOO2-MSL10-GFP, and pOO2MSL10 for in vitro capped RNA production (Maksaev & Haswell, 2012). cRNA for all variants was injected into Xenopus oocytes for expression and characterization as previously described (Maksaev & Haswell, 2015)
We report the functional effect of twelve different point mutations in the mechanosensitive channel MscS-like (MSL)10 from Arabidopsis thaliana

Summary

| INTRODUCTION

The ability to respond to mechanical stimuli is an ancient and intrinsic property of cells (Anishkin, Loukin, Teng, & Kung, 2014; Booth, Miller, Mu€ller, & Lehtovirta-Morley, 2015). To gain additional information about the structure of the channel pore, the mechanism of gating, and how ion flux through the channel is related to its genetic functions, we used mutational analysis and single-channel patch-clamp electrophysiology to identify residues in the presumptive pore-lining domain of MSL10 that are important for channel behavior, including tension sensitivity, conductivity, and stability of the open state. We tested two tension-insensitive mutants for the ability to induce cell death in a previously established transient expression assay These data provide critical information about the structural component of tension-sensitive ion transport and a useful comparison to EcMscS and other MS channels in animals and bacteria. These mutant MSL10 channels provide tools for studying the relationship between tension sensitivity, open-state stability, ion flux, and cell death signaling

| METHODS

| RESULTS

Findings

| DISCUSSION