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The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box.

ABSTRACT: Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a "Jack-In-The-Box" with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure.

SUBMITTER: Malcolm HR

PROVIDER: S-EPMC4259861 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box.

Malcolm Hannah R HR Blount Paul P Maurer Joshua A JA

Biochimica et biophysica acta 20141023 1 Pt A

Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify elev ...[more]

PMID: 25450806

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Project description:Channels from the MscS family are adaptive tension-activated osmolyte release valves that regulate turgor in prokaryotes and volume in plant chloroplasts. The crystal structure of Escherichia coli MscS has provided a starting point for detailed descriptions of its mechanism. However, solved in the absence of the lipid bilayer, this structure may deviate from a native conformation. In this study, we utilized molecular dynamics simulations and a new iterative extrapolated-motion protocol to pack the splayed peripheral TM1 and TM2 transmembrane helices along the central TM3 shaft. This modification restored the tension transmission route between the membrane and the channel gate. We also modeled the structure of the 26-amino acid N-terminal segments that were unresolved in the crystals. The resulting compact conformation, which we believe approximates the closed resting state of MscS, matches the hydrophobic thickness of the lipid bilayer with arginines 46, 54, and 74 facing the polar lipid headgroups. The pore-lining helices in this resting state feature alternative kinks near the conserved G121 instead of the G113 kinks observed in the crystal structure and the transmembrane barrel remains stable in extended molecular dynamics simulations. Further analysis of the dynamics of the pore constriction revealed several moderately asymmetric and largely dehydrated states. Biochemical and patch-clamp experiments with engineered double-cysteine mutants demonstrated cross-linking between predicted adjacent residue pairs, which formed either spontaneously or under moderate oxidation. The L72C-V99C bridge linking more peripheral TM2 to TM3 caused a shift of channel activation to higher pressures. TM3 to TM3 cross-links through the A84C-T93C, S95C-I97C, and A106C-G108C cysteine pairs were shown to lock MscS in a nonconductive state. Normal channel activity in these mutants could be recovered upon disulfide reduction with dithiothreitol. These results confirmed our modeling predictions of a closed MscS channel featuring a TM3 barrel that largely resembles the crystal conformation though with more tightly packed peripheral helices. From this closed-resting conformation, the TM3 helices must expand to allow for channel opening.

Project description:Mechanosensitive channels of small conductance (MscS) are ubiquitous turgor pressure regulators found in many walled cells and some intracellular organelles. Escherichia coli MscS acting as a tension-activated osmolyte release valve shows a nonsaturable conductance (1.2 nS in a 39 mS/cm electrolyte) and weak preference for anions. Pursuing the transition pathways in this channel, we applied the extrapolated motion protocol (cycles of displacements, minimizations, and short simulations) to the previously generated compact resting conformation of MscS. We observed tilting and straightening of the kinked pore-forming TM3 helices during the barrel expansion. Extended all-atom simulations confirmed the stability of the open conformation in the bilayer. A 53 degrees spontaneous axial rotation of TM3s observed after equilibration increased the width and polarity of the pore allowing for stable voltage-independent hydration and presence of both cations and anions throughout the pore. The resultant open state, characterized by a pore 1.6 nm wide, satisfied the experimental conductance and in-plane expansion. Applied transmembrane electric field (+/-100 to +/-200 mV) in simulations produced a flow of both K(+) and Cl(-), with Cl(-) current dominating at higher voltages. Electroosmotic water flux strongly correlated with the chloride current (approximately 8 waters per Cl(-)). The selectivity and rectification were in agreement with the experimental measurements performed in the same range of voltages. Among the charged residues surrounding the pore, only K169 was found to contribute noticeably in the rectification. We conclude that (a) the barrel expansion involving tilting, straightening, and rotation of TM3s provides the geometry and electrostatics that accounts for the conductive properties of the open pore; (b) the observed regimen of ion passage through the pore is similar to electrodiffusion, thus macroscopic estimations closely approximate the experimental and molecular dynamics-simulated conductances; (c) increased interaction of the opposing ionic fluxes at higher voltages may result in selectivities stronger than measured near the reversal potential.

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The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box.

Publications

The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box.

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