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

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.

Stretch-activated ion channels identified in the touch-sensitive structures of carnivorous Droseraceae plants

“…Whereas selectivity for chloride in both mutants remained unchanged, the K579E mutant exhibited smaller single channel currents at positive membrane potentials (Figure 4-figure supplement 3), suggesting that K579 is indeed in the vicinity of the pore. This analysis confirms that the predicted TM6 of FLYC1 is part of the pore-lining region of the channel, consistent with mutagenesis results in MSL10 (Maksaev et al, 2018). These observations are further supported by the cryo-EM (electron microscopy) structure of AtMSL1, which indicates a similar architecture of the last TM of the channel (Deng et al, 2020).…”

The Mechanosensitive Ion Channel MSL10 Potentiates Responses to Cell Swelling in Arabidopsis Seedlings

“…Another possibility is that MSL10's ability to induce cell death, PCD, [Ca 2+ ] cyt transients, ROS accumulation, and gene expression is independent of its ion channel function. Several lines of evidence suggest that MSL10 has a non-conducting function that is mediated through its soluble N-terminal domain: (1) phosphomimetic lesions in the soluble N terminus prevent MSL10 from inducing ROS accumulation and cell death, and phosphodead lesions exacerbate these effects [51,52]; (2) neither type of lesion affects MSL10's mechanosensitive ion channel activity when expressed in oocytes [51]; and (3) MSL10 variants with pore-blocking mutations are able to induce cell death when transiently overexpressed in tobacco cells [93]. As expression of phosphomimetic variants of MSL10 also prevented cell swelling-induced PCD (Figure 6), this function may be independent of ion flux through the MSL10 channel pore.…”

Charged pore-lining residues are required for normal channel kinetics in the eukaryotic mechanosensitive ion channel MSL1

“…MSL2/3 and MSL8 are involved in osmoregulation of chloroplasts and pollen, respectively [28,32,34], much like EcMscS in E. coli cells. However, MSL10 has a cell-death signaling activity that is separable from its MS channel activity [35,36], revealing MSL function beyond maintaining osmotic homeostasis.…”