One face of a transmembrane helix is crucial in mechanosensitive channel gating

Abstract

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an ␣-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.

Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

“…Thus, we focus on subtler changes that cause the channel to tend toward opening. Such rare mutations have been recovered from screens in Escherichia coli for a mechanosensitive channel (24) or in yeast for native (25) and foreign K ϩ channel (26) after single-gene mutagenesis. Among TRP channels, disease or experimental point mutations reported to date all seem to cause reduction or loss of channel activities, including the dominant polycystic kidney disease (PKD) alleles (27) and a dominant-negative construct of TrpV1 (28).…”

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

“…It has been suggested that a Gly residue and its adjacent amino acid residues in the gate play important roles in the gating mechanism in several ion channels. For example, the most conserved Gly 22 residue of E. coli MscL functions as the gate (40,41). When Gly 22 is replaced with hydrophilic residues, the resulting mutant MscL channels become more mechanosensitive.…”

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

“…12 Many studies have been performed using site-directed mutagenesis in order to better understand the structure-function of MscL. [13][14][15][16][17][18][19] One of these studies produced an estimation of the tension-sensing site in MscL using random scanning mutagenesis, where individual hydrophobic AAs facing the lipids were replaced with the hydrophilic AA asparagine to identify any "loss-of-function" mutants lacking mechanosensitivity. 15 As the result, it was found that replacement of one of seven amino residues located at the periplasmic end of the transmembrane helices caused the loss of MscL mechanosensitivity, suggesting that one or some of them may act as a tension sensor in MscL.…”