Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.     

Force Transduction and Lipid Binding in MscL: A Continuum-Molecular Approach

The bacterial mechanosensitive channel MscL, a small protein mainly activated by membrane tension, is a central model system to study the transduction of mechanical stimuli into chemical signals. Mutagenic studies suggest that MscL gating strongly depends on both intra-protein and interfacial lipid-protein interactions. However, there is a gap between this detailed chemical information and current mechanical models of MscL gating. Here, we investigate the MscL bilayer-protein interface through molecular dynamics simulations, and take a combined continuum-molecular approach to connect chemistry and mechanics. We quantify the effect of membrane tension on the forces acting on the surface of the channel, and identify interactions that may be critical in the force transduction between the membrane and MscL. We find that the local stress distribution on the protein surface is largely asymmetric, particularly under tension, with the cytoplasmic side showing significantly larger and more localized forces, which pull the protein radially outward. The molecular interactions that mediate this behavior arise from hydrogen bonds between the electronegative oxygens in the lipid headgroup and a cluster of positively charged lysine residues on the amphipathic S1 domain and the C-terminal end of the second trans-membrane helix. We take advantage of this strong interaction (estimated to be 10–13 kT per lipid) to actuate the channel (by applying forces on protein-bound lipids) and explore its sensitivity to the pulling magnitude and direction. We conclude by highlighting the simple motif that confers MscL with strong anchoring to the bilayer, and its presence in various integral membrane proteins including the human mechanosensitive channel K2P1 and bovine rhodopsin.     

Bilayer-mediated clustering and functional interaction of MscL channels

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.     

New insights of membrane environment effects on MscL channel mechanics from theoretical approaches

The prokaryotic mechanosensitive channel of large conductance (MscL) is a remarkable integral membrane protein. During hypo-osmotic shock, it responses to membrane tension through large conformational changes, that lead to an open state of the pore. The structure of the channel from Mycobacterium tuberculosis has been resolved in the closed state. Numerous experiments have attempted to trap the channel in its open state but they did not succeed in obtaining a structure. A gating mechanism has been proposed based on different experimental data but there is no experimental technique available to follow this process in atomic details. In addition, it has been shown that a decrease of the lipid bilayer thickness lowered MscL activation energy and stabilized a structurally distinct closed channel intermediate. Here, we use atomistic molecular dynamics simulations to investigate the effect of the lipid bilayer thinning on our model of the structure of the Escherichia coli. We thoroughly analyze simulations of the channel embedded in two pre-equilibrated membranes differing by their hydrophobic tail length (DMPE and POPE). The MscL structure remains stable in POPE, whereas a distinct structural state is obtained in DMPE in response to hydrophobic mismatch. This latter is obtained by tilts and kinks of the transmembrane helices, leading to a widening and a diminution of the channel height. Part of these motions is guided by a competition between solvent and lipids for the interaction with the periplasmic loops. We finally conduct a principal component analysis of the simulation and compare anharmonic motions with harmonic ones, previously obtained from a coarse-grained normal mode analysis performed on the same structural model. Significant similarities exist between low-frequency harmonic motions and those observed with essential dynamics in DMPE. In summary, change in membrane thickness permits to accelerate the conformational changes involved in the mechanics of the E. coli channel, providing a closed structural intermediate en route to the open state. These results give clues for better understanding why the channel activation energy is lowered in a thinner membrane.     

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.     

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.     

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.     

Membrane tension, lipid adaptation, conformational changes, and energetics in MscL gating

This study aims to explore gating mechanisms of mechanosensitive channels in terms of membrane tension, membrane adaptation, protein conformation, and energetics. The large conductance mechanosensitive channel from Mycobacterium tuberculosis (Tb-MscL) is used as a model system; Tb-MscL acts as a safety valve by releasing small osmolytes through the channel opening under extreme hypoosmotic conditions. Based on the assumption that the channel gating involves tilting of the transmembrane (TM) helices, we have performed free energy simulations of Tb-MscL as a function of TM helix tilt angle in a dimyristoylphosphatidylcholine bilayer. Based on the change in system dimensions, TM helix tilting is shown to be essentially equivalent to applying an excess surface tension to the membrane, causing channel expansion, lipid adaptation, and membrane thinning. Such equivalence is further corroborated by the observation that the free energy cost of Tb-MscL channel expansion is comparable to the work done by the excess surface tension. Tb-MscL TM helix tilting results in an expanded water-conducting channel of an outer dimension similar to the proposed fully open MscL structure. The free energy decomposition indicates a possible expansion mechanism in which tilting and expanding of TM2 facilitates the iris-like motion of TM1, producing an expanded Tb-MscL.     

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.     

Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channel's mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channel's funnel impairs the function of MscL.     

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.     

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.     

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.     

Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups

MscL is a mechanosensitive channel of large conductance that serves as an "emergency relief valve", protecting bacteria from acute hypoosmotic stress. Although it is well-accepted that the MscL protein and an adequate membrane matrix are necessary and sufficient for the function of this channel, the exact role of the membrane has yet to be elucidated. Here, we address the role of the membrane matrix through in vitro reconstitution of the MscL protein in defined lipid bilayers. We have applied Laplace's law to visualized membrane patches where we can measure patch curvature as described in previous studies. Here, by comparing patches with different curvatures, we demonstrate that the MscL channel senses tension within the membrane and that the pressure across it plays no detectable role as a stimulus. In addition, gating only occurs when the smallest radius of curvature is nearly achieved, suggesting that the lateral tension rather than membrane curvature is the important biophysical parameter. Finally, we have examined the contribution of specific headgroups by measuring their effect on the membrane tension required to gate the channel. We have found that the addition of neither anionic nor endogenous lipids to a non-native membrane effected a leftward shift in the activation curve. In fact, the major endogenous lipid of the Escherichia coli membrane, phosphatidylethanolamine, led to a channel activity at a higher tension threshold, suggesting that this lipid effects altered activity through changes in the biophysical properties of the membrane, rather than through an MscL-specific interaction.     

Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating

In mechanosensitive (MS) channels, gating is initiated by changes in intra-bilayer pressure profiles originating from bilayer deformation. Here we evaluated two physical mechanisms as triggers of MS channel gating: the energetic cost of protein-bilayer hydrophobic mismatches and the geometric consequences of bilayer intrinsic curvature. Structural changes in the Escherichia coli large MS channel (MscL) were studied under nominally zero transbilayer pressures using both patch clamp and EPR spectroscopic approaches. Changes in membrane intrinsic curvature induced by the external addition of lysophosphatidylcholine (LPC) generated massive spectroscopic changes in the narrow constriction that forms the channel 'gate', trapping the channel in the fully open state. Hydrophobic mismatch alone was unable to open the channel, but decreasing bilayer thickness lowered MscL activation energy, stabilizing a structurally distinct closed channel intermediate. We propose that the mechanism of mechanotransduction in MS channels is defined by both local and global asymmetries in the transbilayer pressure profile at the lipid-protein interface.     

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.     

Mechanosensitive channels in archaea

The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologues in Archaea, the third domain of the phylogenetic tree. Two types of MS channels have been identified in the cell membranes of Haloferax volcanii using the patch clamp technique. Recently MS channels were identified and cloned from two archaeal species occupying different environmental habitats. These studies demonstrate that archaeal MS channels share structural and functional homology with bacterial MS channels. The mechanical force transmitted via the lipid bilayer alone activates all to date known prokaryotic MS channels. This implies the existence of a common gating mechanism for bacterial as well as archaeal MS channels according to the bilayer model. Based on recent evidence that the bilayer model also applies to eukaryotic MS channels, mechanosensory transduction probably originated along with the appearance of the first life forms according to simple biophysical principles. In support of this hypothesis the phylogenetic analysis revealed that prokaryotic MS channels of large and small conductance originated from a common ancestral molecule resembling the bacterial MscL channel protein. Furthemore, bacterial and archaeal MS channels share common structural motifs with eukaryotic channels of diverse function indicating the importance of identified structures to the gating mechanism of this family of channels. The comparative approach used throughout this review should contribute towards understanding of the evolution and molecular basis of mechanosensory transduction in general.     

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.     

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.