Lipid-protein interaction of the MscS mechanosensitive channel examined by scanning mutagenesis

Abstract

The mechanosensitive channel of small conductance (MscS) is a bacterial mechanosensitive channel that opens in response to rapid hypoosmotic stress. Since MscS can be opened solely by membrane stretch without help from any accessory protein, the lipid-protein interface must play a crucial role in sensing membrane tension. In this study, the hydrophobic residues in the lipid-protein interface were substituted one by one with a hydrophilic amino acid, asparagine, to modify the interaction between the protein and the lipid. Function of the mutant MscSs was examined by patch-clamp and hypoosmotic shock experiments. An increase in the gating threshold and a decrease in the viability on hypoosmotic shock were observed when the hydrophobic residues near either end of the first or the second transmembrane helix (TM1 or TM2) were replaced with asparagine. This observation indicates that the lipid-protein interaction at the ends of both helices (TM1 and TM2) is essential to MscS function.

FIGURE 1

The residues used for asparagine substitution displayed on the crystal structure of MscS. The residues in TM1 and TM2 are shown in space fill model and the loop connecting TM1 and TM2 is shown in ribbon model. The residues in a single subunit are colored arbitrarily except for hydrophilic residues, which are displayed in blue. The residues that are not visible from the outside are in brackets.

FIGURE 2

Characterization of the wild-type and mutant MscS. (A–C) Channel current through the wild-type (A), I48N (B), and L55N MscS (C). In each panel, the membrane current (top) and the negative pressure applied through patch pipette (bottom) are shown. The first opening of MscS and MscL upon continuously increasing suction is indicated by arrowheads and arrows, respectively. Pipette potential was +15 mV. (D) Threshold for MscS gating as determined by the patch-clamp (mean ± SE). The asterisks in D and E indicate that the threshold is significantly different from wild-type (p < 0.05 by t-test). ND, not determined. (E) Effects of hypoosmotic shock on cells expressing mutant MscS (mean ± SE).

FIGURE 3

Summary of the impact (severity of function loss) of asparagine substitution. (A) Survival rate plotted against the gating thresholds. The density of blue and red increases with the decrease and increase in the gating threshold from the wild-type MscS, respectively. (B) Changes in the side chain of the “tight” mutation sites. (C, D) The impact of asparagine substitution shown on net diagram (C) and structural model (D) of TM1 and TM2. (C) Gain-of-function (GOF) mutants are shown in black (V40D is from Okada et al. (9) and I78N is from this experiment). The residues that were not subjected to mutagenesis are in gray. Thick circles indicate that the residues are conserved among MscS from various origins. Parts of each helix facing the lipid in the crystal structure are shown in orange.

FIGURE 4

Effect of combining the mutations that alter MscS stiffer than the wild-type MscS. (A) Survival rate of the double mutants (mean ± SE). The asterisks indicate that the survival rate is significantly different from the corresponding single mutants (p < 0.05). (B) Western blot against the histidine tag attached to the carboxy terminus of MscS. (C, D) Patch clamp experiment on the cells expressing A51N/F68N (C) and I37N/L86N MscS (D). The insets show the magnification of the MscL traces indicated by arrows. Pipette potential was +15 mV.

FIGURE 5

Diagram of the proposed movement of transmembrane helices. Two subunits, each consisting of TM1, TM2, and the membrane embedded part of TM3 (boxes), are shown. Semicircles in the boxes represent the residues for the “tight” mutations in TM1 and TM2. The semicircles are shown as open when a residue within the site is substituted with asparagine. Thin lines at the top and the bottom are the periplasmic and cytoplasmic surface of the membrane, respectively. (A) Closed structure of MscS. (B) Channel opening is brought about by parallel movement of TM1 and TM2. (C, D) When two asparagine substitutions were set to cytoplasmic ends of TM1 and TM2 (C; e.g., A51N/F68N) or to the periplasmic ends (D; e.g., I37N/l86N) tilting of TM1 and TM2 occurs by an unbalanced expansion at both ends. In this case, channel opening does not occur.