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

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: From tension sensing to channel opening

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Engineering covalent oligomers of the mechanosensitive channel of large conductance from Escherichia coli with native conductance and gating characteristics

To obtain a gene construct for making single substitutions per channel and to determine the quaternary structure of the mechanosensitive channel MscL from Escherichia coli, covalent oligomers (monomer to hexamer) were engineered by gene fusion; up to six copies of the mscL gene were fused in tandem. All the multimeric tandem constructs yielded functional channels with wild-type conductance and dwell times. Importantly, only the covalent pentamer opened at the same relative pressure (compared to the pressure required to open MscS) as the wild-type MscL channel. The in vivo data strongly suggest that pentameric MscL represents the functional state of the channel.     

Disulfide trapping the mechanosensitive channel MscL into a gating-transition state

The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock, and is to date the best characterized mechanosensitive channel. The N-terminal region of the protein has been shown to be critical for function by random, site-directed, and deletion mutagenesis, yet is structurally poorly understood. One model proposes that the extreme N-termini form a cluster of amphipathic helices that serves as a cytoplasmic second gate, separated from the pore-forming transmembrane domain by a "linker". Here, we have utilized cysteine trapping of single-cysteine mutated channels to determine the proximity, within the homopentameric complex, of residues within and just peripheral to this proposed linker. Our results indicate that all residues in this region can form disulfide bridges, and that the percentage of dimers increases when the channel is gated in vivo. Functional studies suggest that oxidation traps one of these mutated channels, N15C, into a gating-transition state that retains the capacity to obtain both fully open and closed states. The data are not easily explained by current models for the smooth transition from closed-to-open states, but predict that an asymmetric movement of one or more of the subunits commonly occurs upon gating.     

Anionic phospholipids affect the rate and extent of flux through the mechanosensitive channel of large conductance MscL

The mechanosensitive channel of large conductance MscL from Escherichia coli has been reconstituted into sealed vesicles, and the effects of lipid structure on the flux of the fluorescent molecule calcein through the open channel have been studied. The channel was opened by reaction of the G22C mutant of MscL with the reagent [2-(triethylammonium)ethyl]methanethiosulfonate (MTSET) which introduces five positive charges within the pore constriction. Flux through the channel was small when the lipid was phosphatidylcholine, but addition of the anionic lipids phosphatidylglycerol, phosphatidic acid, or cardiolipin up to 50 mol % resulted in increases in the amplitudes and rates of release of calcein. Similar effects were seen when either wild-type MscL or the G22C mutant was opened by osmotic pressure difference; rates of release of calcein were very slow in the absence of anionic lipid but increased with increasing concentrations of phosphatidylglycerol to 50 mol %. The observed partial release of trapped calcein following activation of MscL was attributed to the formation of a long-lived subconductance state of MscL following channel opening. Effects of anionic lipid were attributed to an increase in the rate of the transition from closed to fully open state and to a decrease in the rate of the transition from the fully open state to the subconductance state. Higher concentrations of anionic lipid led to a decrease in the rate and amplitude of release of calcein, possibly due to a decreased rate of flux through the open channel. In mixtures with anionic lipids, phosphatidylethanolamine resulted in lower rates and amplitude of release than phosphatidylcholine.     

Hidden Markov analysis of mechanosensitive ion channel gating

Patch clamp data from the large conductance mechanosensitive channel (MscL) in E. coli was studied with the aim of developing a strategy for statistical analysis based on hidden Markov models (HMMs) and determining the number of conductance levels of the channel, together with mean current, mean dwell time and equilibrium probability of occupancy for each level. The models incorporated state-dependent white noise and moving average adjustment for filtering, with maximum likelihood parameter estimates obtained using an EM (expectation-maximisation) based iteration. Adjustment for filtering was included as it could be expected that the electronic filter used in recording would have a major effect on obviously brief intermediate conductance level sojourns. Preliminary data analysis revealed that the brevity of intermediate level sojourns caused difficulties in assignment of data points to levels as a result of over-estimation of noise variances. When reasonable constraints were placed on these variances using the better determined noise variances for the closed and fully open levels, idealisation anomalies were eliminated. Nevertheless, simulations suggested that mean sojourn times for the intermediate levels were still considerably over-estimated, and that recording bandwidth was a major limitation; improved results were obtained with higher bandwidth data (10 kHz sampled at 25 kHz). The simplest model consistent with these data had four open conductance levels, intermediate levels being approximately 20%, 51% and 74% of fully open. The mean lifetime at the fully open level was about 1 ms; estimates for the three intermediate levels were 54-92 micros, probably still over-estimates.     

Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance,MscL: implications for channel gating and evolutionary design

Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function. Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified. Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function. The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer. The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels.     

Investigating lipid composition effects on the mechanosensitive channel of large conductance (MscL) using molecular dynamics simulations

Previous experimental work has shown that the functional properties of the mechanosensitive channel of large conductance (MscL) are affected by variations in lipid composition. Here, we utilize molecular dynamics simulations of Mycobacterium tuberculosis MscL to investigate such lipid composition effects on a molecular level. In particular, two sets of simulations were performed. In the first, trajectories using lipids with different headgroups (phosphatidylcholine and phosphatidylethanolamine) were compared. Protein-lipid interactions were clearly altered by the headgroup changes, leading to conformational differences in the C-terminal region of M. tuberculosis MscL. In the second set of simulations, lipid tails were gradually shortened, thinning the membrane over a molecular dynamics trajectory. These simulations showed evidence of hydrophobic matching between MscL and the lipid membrane, as previously proposed. For all simulations, protein-lipid interaction energies in the second transmembrane region were correlated to mutagenic data, emphasizing the importance of lipid interactions for proper MscL function.     

On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.     

Voltage-induced gating of the mechanosensitive MscL ion channel reconstituted in a tethered lipid bilayer membrane

The mechanosensitive (MS) ion channel is gated by changes in bilayer deformation. It is functional without the presence of any other proteins and gating of the channel has been successfully achieved using conventional patch clamping techniques where a voltage has been applied together with a pressure over the membrane. Here, we have for the first time analyzed the large conducting (MscL) channel in a supported membrane using only an external electrical field. This was made possible using a newly developed technique utilizing a tethered lipid bilayer membrane (tBLM), which is part of an engineered microelectronic array chip. Single ion channel activity characteristic for MscL was obtained, albeit with lower conductivity. The ion channel was gated using solely a transmembrane potential of 300 mV. Computations demonstrate that this amount of membrane potential induces a membrane tension of 12 dyn/cm, equivalent to that calculated to gate the channel in patch clamp from pressure-induced stretching of the bilayer. These results strengthen the supposition that the MscL ion channel gates in response to stress in the lipid membrane rather than pressure across it. Furthermore, these findings illustrate the possibility of using the MscL as a release valve for engineered membrane devices; one step closer to mimicking the true function of the living cell.     

Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method

The bacterial mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance have functionally been reconstituted into giant unilamellar liposomes (GUVs) using an improved reconstitution method in the presence of sucrose. This method gives significant time savings (preparation times as little as 6 h) compared to the classical method of protein reconstitution which uses a dehydration/rehydration (D/R) procedure (minimum 2 days preparation time). Moreover, it represents the first highly reproducible method for functional reconstitution of MscS as well as MscS/MscL co-reconstitution. This novel procedure has the potential to be used for studies of other ion channels by liposome reconstitution.     

Modulation of channel activity and gadolinium block of MscL by static magnetic fields

The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd³⁺ ions from the membrane bilayer and thus remove the MscL channel block.     

Electromechanical coupling model of gating the large mechanosensitive ion channel (MscL) of Escherichia coli by mechanical force.

We have developed a theoretical electromechanical coupling (EMC) model of gating of the large-conductance mechanosensitive ion channel (MscL). The model presents the first attempt to explain the pressure-dependent transitions between the closed and open channel conformations on a molecular level by assuming 1) a homohexameric structural model of the channel, 2) electrostatic interactions between various domains of the homohexamer, 3) structural flexibility of the N-terminal portion of the monomer, and 4) mechanically and electrostatically induced displacement of the N-terminal domain relative to other structural domains of the protein. In the EMC model, 12 membrane-spanning alpha-helices (six each of the M1 and M2 transmembrane domains of the MscL monomer), are envisaged to line the channel pore with a diameter of 40 A, whereas the N- and C-termini are oriented toward each other inside the pore when the channel is closed. The model proposes that stretching the membrane bilayer by mechanical force causes the monomers to be pulled away from and slightly tilted toward each other. This relative movement of alpha-helices could serve as a trigger to initiate a "swing-like" motion of the N-terminus around the glycine residue G14 that may act as a pivot. The analysis of the attractive and repulsive coulomb forces between all domains of the channel homohexamer suggested that an inclination angle of approximately 3.0 degrees - 4.1 degrees between the oppositely oriented channel monomers should suffice for the N-terminus to turn away from other domains causing the channel to open. According to the EMC model the minimal free energy change, deltaG, that could initiate the opening of the channel was 2 kT. Also, the model predicted that the negative pressure required for channel open probability, Po = 0.5, should be between 50 and 80 mmHg. These values were in a good agreement with the experimentally estimated pressures of 60-70 mmHg obtained with the MscL reconstituted in liposomes. Furthermore, consistent with a notion that the N-terminus may present a mechanosensitive structural element providing a mechanism to open the MscL by mechanical force, the model provides a simple explanation for the variations in pressure sensitivity observed with several MscL mutants having either deletions or substitutions in N- or C-terminus, or site-directed mutations in the S2-S3 loop.     

Structure and function of the bacterial mechanosensitive channel of large conductance.

Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.     

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: Mutants with altered channel gating and pressure sensitivity

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic α-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning α-helix. Received: 25 September 1996/Revised: 21 November 1996     

Defining the physical gate of a mechanosensitive channel, MscL, by engineering metal-binding sites

The mechanosensitive channel of large conductance, MscL, of Escherichia coli is one of the best-studied mechanosensitive proteins. Although the structure of the closed or "nearly-closed" state of the Mycobacterium tuberculosis ortholog has been solved and mechanisms of gating have been proposed, the transition from the closed to the open states remains controversial. Here, we probe the relative position of specific residues predicted to line the pore of MscL in either the closed state or during the closed-to-open transition by engineering single-site histidine substitutions and assessing the ability of Ni2+, Cd2+ or Zn2+ ions to affect channel activity. All residues predicted to be within the pore led to a change in channel threshold pressure, although the direction and extent of this change were dependent upon the mutation and metal used. One of the MscL mutants, L19H, exhibited gating that was inhibited by Cd2+ but stimulated by Ni2+, suggesting that these metals bind to and influence different states of the channel. Together, the results derived from this study support the hypotheses that the crystal structure depicts a "nearly closed" rather than a "fully closed" state of MscL, and that a clockwise rotation of transmembrane domain 1 occurs early in the gating process.     

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL–lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL–lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of β-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The ΔmscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase ΔmscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, ΔmscL cells had become resistant to a 0.9 M osmotic downshift.     

Intragenic suppression of gain-of-function mutations in the Escherichia coli mechanosensitive channel, MscL

Mechanosensitive channels play an important role in protecting bacterial cells from osmotic downshock by serving as biological 'pressure release valves'. One of these channels, MscL, is found throughout the bacterial kingdom, but has been most studied in Escherichia coli. The E. coli MscL is a 136-amino-acid protein organized as a homopentamer with each subunit containing two transmembrane segments. Previous studies have shown that several residues, including V23 and G26, are essential for normal function of MscL; very severe gain-of-function phenotypes in which cell growth slows or is arrested can result from residue substitutions at these positions. Through random mutagenesis and growth selection, we have generated intragenic suppressors of the V23A and G26S mutations. The suppressor mutants have been characterized by growth phenotype, Western blot and patch clamp. Most of the mutations that render phenotypic suppression are located in the transmembrane domains with additional sites lying in the periplasmic loop. In contrast, only one mutation is found in the amino-terminal S1 domain, and none is found within the carboxyl-terminal domain. Not only have these findings revealed functional domains and subdomains critical for MscL function, but they also predict a pair of residues that interact directly during channel opening.     

MscS, the bacterial mechanosensitive channel of small conductance

The mechanosensitive channel of small conductance, MscS, is one of the most extensively studied MS channels to date. Past and present research involves the discovery of its physiological role as an emergency valve in prokaryotes up to detailed investigations of its conductive properties and gating mechanism. In this review, we summarize the findings on its structure and function obtained by experimental and theoretical approaches. A special focus is given to its pharmacology, since various compounds have been shown to affect the activity of this channel. These compounds have particularly been helpful for understanding the interaction of MscS with the lipid bilayer, as well as recognizing the potential of this channel as a target for novel types of antibiotics.