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.     

Gating of MscL studied by steered molecular dynamics

Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel. A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface. The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation. A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening. The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices. The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL. Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening.     

Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity.

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other common amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tuberculosis (, Science. 282:2220-2226), Gly(22) is buried within the constriction that closes the pore. Substituting Gly(22) with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

Structural determinants of MscL gating studied by molecular dynamics simulations

The mechanosensitive channel of large conductance (MscL) in prokaryotes plays a crucial role in exocytosis as well as in the response to osmotic downshock. The channel can be gated by tension in the membrane bilayer. The determination of functionally important residues in MscL, patch-clamp studies of pressure-conductance relationships, and the recently elucidated crystal structure of MscL from Mycobacterium tuberculosis have guided the search for the mechanism of MscL gating. Here, we present a molecular dynamics study of the MscL protein embedded in a fully hydrated POPC bilayer. Simulations totaling 3 ns in length were carried out under conditions of constant temperature and pressure using periodic boundary conditions and full electrostatics. The protein remained in the closed state corresponding to the crystal structure, as evidenced by its impermeability to water. Analysis of equilibrium fluctuations showed that the protein was least mobile in the narrowest part of the channel. The gating process was investigated through simulations of the bare protein under conditions of constant surface tension. Under a range of conditions, the transmembrane helices flattened as the pore widened. Implications for the gating mechanism in light of these and experimental results are discussed.     

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.     

Gain-of-function mutations reveal expanded intermediate states and a sequential action of two gates in MscL

The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) --> low-conducting substate (S) --> open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C-->S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area ((Delta)A) and energy ((Delta)E) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger (Delta)E associated with the second (S-->O) subtransition, but a (Delta)A similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (k(off)) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C-->S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled "leaky" conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1-M1 linkers result in a much higher probability of reverse O-->S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S-->O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.     

Mechanosensitive channel gating transitions resolved by functional changes upon pore modification

The mechanosensitive channel of large conductance acts as a biological "emergency release valve" that protects bacterial cells from hypoosmotic stress. Although structural and functional studies and molecular dynamic simulations of this channel have led to several models for the structural transitions that occur in the gating process, inconsistencies linger and details are lacking. A previous study, using a method coined as the "in vivo SCAM", identified several residues in the channel pore that were exposed to the aqueous environment in the closed and opening conformations. Briefly, the sulfhydryl reagent MTSET was allowed to react, in the presence or absence of hypoosmotic shock, with cells expressing mechanosensitive channel of large conductance channels that contained cysteine substitutions; channel dysfunction was assessed solely by cell viability. Here we evaluate the MTSET-induced functional modifications to these mechanosensitive channel activities by measuring single channel recordings. The observed changes in residue availability in different states, as well as channel kinetics and sensitivity, have allowed us to elucidate the microenvironment encountered for a number of pore residues, thus testing many aspects of previous models and giving a higher resolution of the pore domain and the structural transitions it undergoes from the closed to open state.     

On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface

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. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.     

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.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

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.     

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.     

Gating mechanism of mechanosensitive channel of large conductance: a coupled continuum mechanical-continuum solvation approach

Gating transition of the mechanosensitive channel of large conductance (MscL) represents a good example of important biological processes that are difficult to describe using atomistic simulations due to the large (submicron) length scale and long (millisecond) time scale. Here we develop a novel computational framework that tightly couples continuum mechanics with continuum solvation models to study the detailed gating behavior of E. coli-MscL. The components of protein molecules are modeled by continuum elements that properly describe their shape, material properties and physicochemical features (e.g., charge distribution). The lipid membrane is modeled as a three-layer material in which the lipid head group and tail regions are treated separately, taking into account the fact that fluidic lipid bilayers do not bear shear stress. Coupling between mechanical and chemical responses of the channel is realized by an iterative integration of continuum mechanics (CM) modeling and continuum solvation (CS) computation. Compared to previous continuum mechanics studies, the present model is capable of capturing the most essential features of the gating process in a much more realistic fashion: due mainly to the apolar solvation contribution, the membrane tension for full opening of MscL is reduced substantially to the experimental measured range. Moreover, the pore size stabilizes constantly during gating because of the intricate interactions of the multiple components of the system, implying the mechanism for sub-conducting states of MscL gating. A significant fraction (\(\sim \)2/3) of the gating membrane strain is required to reach the first sub-conducting state of our model, which is featured with a relative conductance of 0.115 to the fully opened state. These trends agree well with experimental observations. We anticipate that the coupled CM/CS modeling framework is uniquely suited for the analysis of many biomolecules and their assemblies under external mechanical stimuli.     

Structural models of the MscL gating mechanism.

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.     

Chemically charging the pore constriction opens the mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that protects the cell from osmotic downshock. We have previously shown that substitution of a residue that resides within the channel pore constriction, MscL's Gly-22, with all other 19 amino acids affects channel gating according to the hydrophobicity of the substitution (). Here, we first make a mild substitution, G22C, and then attach methanethiosulfonate (MTS) reagents to the cysteine under patch clamp. Binding MTS reagents that are positively charged ([2-(trimethylammonium)ethyl] methanethiosulfonate and 2-aminoethyl methanethiosulfonate) or negatively charged (sodium (2-sulfonatoethyl)methanethiosulfonate) causes MscL to gate spontaneously, even when no tension is applied. In contrast, the polar 2-hydroxyethyl methanethiosulfonate halves the threshold, and the hydrophobic methyl methanethiolsulfonate increases the threshold. These observations indicate that residue 22 is in a hydrophobic environment before gating and in a hydrophilic environment during opening to a substate, a finding consistent with our previous study. In addition, we have found that cysteine 22 is accessible to reagents from the cytoplasmic side only when the channel is opened whereas it is accessible from the periplasmic side even in the closed state. These results support the view that exposure of hydrophobic surfaces to a hydrophilic environment during channel opening serves as the barrier to gating.     

Purification and functional reconstitution of N- and C-halves of the MscL channel

MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100 mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity.     

The effect of local bending on gating of MscL using a representative volume element and finite element simulation

Many physiological processes such as cell division, endocytosis and exocytosis cause severe local curvature of the cell membrane. Local curvature has been shown experimentally to modulate numerous mechanosensitive (MS) ion channels. In order to quantify the effects of local curvature we introduced a coarse grain representative volume element for the bacterial mechanosensitive ion channel of large conductance (MscL) using continuum elasticity. Our model is designed to be consistent with the channel conformation in the closed and open states to capture its major continuum rheological behavior in response to the local membrane curvature. Herein we show that change in the local curvature of the lipid bilayer can modulate MscL activity considerably by changing both bilayer thickness and lateral pressure profile. Intriguingly, although bending in any direction results in almost the same free-energy cost, inward (cytoplasmic) bending favors channel opening, whereas outward (periplasmic) bending facilitates closing of the narrowest part of the MscL pore. This quantitative study using MscL as a model channel may have wide reaching consequences for the effect of local curvature on the physiological function of other types of prokaryotic and eukaryotic membrane proteins.