Activation Of MscL Research Articles

A novel force transduction pathway from a tension sensor to the gate in the mechano-gating of MscL channel.

The bacterial mechanosensitive channel of large conductance MscL is activated exclusively by increased tension in the membrane bilayer. Despite many proposed models for MscL opening, its precise mechano-gating mechanism, particularly how the received force at the tension sensor transmits to the gate remains incomplete. Previous studies have shown that along with amphipathic N-terminus located near the cytoplasmic surface of the membrane, Phe78 residue near the outer surface also acts as a "tension sensor," while Gly22 is a central constituent of the "hydrophobic gate." Present study focused on elucidating the force transmission mechanism from the sensor Phe78 in the outer transmembrane helix (TM2) to the gate in the inner transmembrane helix (TM1) of MscL by applying the patch clamp and molecular dynamics (MD) simulations to the wild type MscL channel and its single mutants at the sensor (F78N), the gate (G22N) and their combination (G22N/F78N) double mutant. F78N MscL resulted in a severe loss-of-function, while G22N MscL caused a gain-of-function channel exhibiting spontaneous openings at the resting membrane tension. We initially speculated that the spontaneous opening in G22N mutant might occur without tension acting on Phe78 residue. To test this hypothesis, we examined the (G22N/F78N) double mutant, which unexpectedly exhibited neither spontaneous activity nor activity by a relatively high membrane tension. To understand the underlying mechanism, we conducted MD simulations and analyzed the force transduction pathway. Results showed that the mutation at the tension sensor (F78N) in TM2 caused decreased interaction of this residue not only with lipids, but also with a group of amino acids (Ile32-Leu36-Ile40) in the neighboring TM1 helix, which resulted in an inefficient force transmission to the gate-constituting amino acids on TM1. This change also induced a slight tilting of TM1 towards the membrane plane and decreased the size of the channel pore at the gate, which seems to be the major mechanism for the inhibition of spontaneous opening of the double mutant channel. More importantly, the newly identified interaction between the TM2 (Phe78) and adjacent TM1 (Ile32-Leu36-Ile40) helices seems to be an essential force transmitting mechanism for the stretch-dependent activation of MscL given that substitution of any one of these four amino acids with Asn resulted in severe loss-of-function MscL as reported in our previous work.

Elucidating the molecular basis of spontaneous activation in an engineered mechanosensitive channel.

Mechanosensitive channel of large conductance (MscL) protein detect and respond to changes in the pressure profile of cellular membranes and transduce the mechanical energy into electrical and/or chemical signals. MscL can be activated using ultrasonic or chemical activation methods to improve the absorption of medicines and bioactive compounds into cells. However, by re-engineering the MscL, chemical signals such as pH change can trigger the channel's activation. This paper elucidates the activation mechanism of an engineered and wild-type (WT) MscL at an atomic level through a combination of equilibrium and non-equilibrium (NE) molecular dynamics (MD) simulations, the String method with swarms of trajectories (SMwST), and Expectation maximization (EM) methods. Compared to the WT and engineered MscL activation processes, it suggests that the two systems are likely associated with different active states and different transition pathways. These findings indicate that (1) periplasmic loops play a key role in the activation process of MscL; (2) the loss of various backbone-backbone hydrogen bonds and salt bridge interactions in the engineered MscL channel causes the spontaneous opening of the channel; and (3) the most significant interactions that were lost during the activation process were those between the transmembrane helices 1 and 2 in the engineered MscL channel. In this work, the orientation-based biasing approach for producing and optimizing an open MscL model is a promising way to systematically characterize unknown protein functional states and to research the activation processes in ion channels. This work makes it possible to study ways to design pH-triggered channel-functionalized liposomes for drug delivery.

Cyclodextrins for structural and functional studies of mechanosensitive channels

Mechanosensitive (MS) channels that are activated by the ‘force-from-lipids’ (FFL) principle rest in the membrane in a closed state but open a transmembrane pore in response to changes in the transmembrane pressure profile. The molecular implementations of the FFL principle vary widely between different MS channel families. The function of MS channels is often studied by patch-clamp electrophysiology, in which mechanical force or amphipathic molecules are used to activate the channels. Structural studies of MS channels in states other than the closed resting state typically relied on the use of mutant channels. Cyclodextrins (CDs) were recently introduced as a relatively easy and convenient approach to generate membrane tension. The principle is that CDs chelate hydrophobic molecules and can remove lipids from membranes, thus forcing the remaining lipids to cover more surface area and creating tension for membrane proteins residing in the membranes. CDs can be used to study the structure of MS channels in a membrane under tension by using single-particle cryo-electron microscopy to image the channels in nanodiscs after incubation with CDs as well as to characterize the function of MS channels by using patch-clamp electrophysiology to record the effect of CDs on channels inserted into membrane patches excised from proteoliposomes. Importantly, because incubation of membrane patches with CDs results in the activation of MscL, an MS channel that opens only shortly before membrane rupture, CD-mediated lipid removal appears to generate sufficient force to open most if not all types of MS channels that follow the FFL principle.

Mechanical Activation of MscL Revealed by a Locally Distributed Tension Molecular Dynamics Approach

Membrane tension perceived by mechanosensitive (MS) proteins mediates cellular responses to mechanical stimuli and osmotic stresses, and it also guides multiple biological functions including cardiovascular control and development. In bacteria, MS channels function as tension-activated pores limiting excessive turgor pressure, with MS channel of large conductance (MscL) acting as an emergency release valve preventing cell lysis. Previous attempts to simulate gating transitions in MscL by either directly applying steering forces to the protein or by increasing the whole-system tension were not fully successful and often disrupted the integrity of the system. We present a novel, to our knowledge, locally distributed tension molecular dynamics (LDT-MD) simulation method that allows application of forces continuously distributed among lipids surrounding the channel using a specially constructed collective variable. We report reproducible and reversible transitions of MscL to the open state with measured parameters of lateral expansion and conductivity that exactly satisfy experimental values. The LDT-MD method enables exploration of the MscL-gating process with different pulling velocities and variable tension asymmetry between the inner and outer membrane leaflets. We use LDT-MD in combination with well-tempered metadynamics to reconstruct the tension-dependent free-energy landscape for the opening transition in MscL. The flexible definition of the LDT collective variable allows general application of our method to study mechanical activation of any membrane-embedded protein.

Novel MscL agonists that allow multiple antibiotics cytoplasmic access activate the channel through a common binding site.

The antibiotic resistance crisis is becoming dire, yet in the past several years few potential antibiotics or adjuvants with novel modes of action have been identified. The bacterial mechanosensitive channel of large conductance, MscL, found in the majority of bacterial species, including pathogens, normally functions as an emergency release valve, sensing membrane tension upon low-osmotic stress and discharging cytoplasmic solutes before cell lysis. Opening the huge ~30Å diameter pore of MscL inappropriately is detrimental to the cell, allowing solutes from and even passage of drugs into to cytoplasm. Thus, MscL is a potential novel drug target. However, there are no known natural agonists, and small compounds that modulate MscL activity are just now being identified. Here we describe a small compound, K05, that specifically modulates MscL activity and we compare results with those obtained for the recently characterized MscL agonist 011A. While the structure of K05 only vaguely resembles 011A, many of the findings, including the binding pocket, are similar. On the other hand, both in vivo and molecular dynamic simulations indicate that the two compounds modulate MscL activity in significantly different ways.

Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms.

The bacterial mechanosensitive channel of large conductance (MscL) normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. Opening the large pore of MscL inappropriately is detrimental to the cell, and thus it has been speculated to be a potential antibiotic target. Although MscL is one of the best studied mechanosensitive channels, no chemical that influenced bacterial growth by modulating MscL is known. We therefore used a high-throughput screen to identify compounds that slowed growth in an MscL-dependent manner. We characterized 2 novel sulfonamide compounds identified in the screen. We demonstrated that, although both increase MscL gating, one of these compounds does not work through the folate pathway, as other antimicrobial sulfonamides; indeed, the sulfonamide portion of the compound is not needed for activity. The only mode of action appears to be MscL activation. The binding pocket is where an α-helix runs along the cytoplasmic membrane and interacts with a neighboring subunit; analogous motifs have been observed in several prokaryotic and eukaryotic channels. The data not only demonstrate that MscL is a viable antibiotic target, but also give insight into the gating mechanisms of MscL, and they may have implications for developing agonists for other channels.-Wray, R., Iscla, I., Kovacs, Z., Wang, J., Blount, P. Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms.

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers

The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.

Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension.

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1–GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.

Magnetically triggered smart liposomes

Event Abstract Back to Event Magnetically triggered smart liposomes Md Shahriar Hossain1*, Boris Martinac2*, Jung Ho Kim1*, M Azam Ali3*, Mislav Mustapic1*, Yoshitaka Nakayama2* and Joseph Horvat1* 1 Institute for Superconducting and Electronic Materials, University of Wollongong, Science and Engineering, Australia 2 Victor Chang Cardiac Research Institute, Mechano Biology Laboratory, Australia 3 University of Otago, Department of Applied Science, New Zealand Introduction: The magnetic properties of superparamagnetic nanoparticles have been the subject of research for many years. Within the last decade there has been a rapidly growing interest for magnetic nanoparticles motivated by numerous existing and expected applications in biomedicine, like separation of magnetically tagged cells, targeted drug or radionuclide transport into the cells or tissues, contrast improvement in diagnostic MRI and magnetic hyperthermia (local destruction of tumour tissues by inductive heating), amongst many others. The use of magnetic nanoparticles as a drug delivering system is still defined by its biocompatibility and selective targeting to the desired cell or tissue under the guidance of external magnetic field. Advances in current technologies and the development of magnetic nanoparticles as drug delivery systems to deliver drugs to tumor hypoxic zones have fast-tracked in the past decade and led to the development of various magnetic nano-formulations such as liposomes, metallic/nonmetallic, and polymeric nanoparticles. In this study, we have developed superparamagnetic CoFe2O4 nanoparticles for binding to MscL. We show their lack of toxicity on a human cell culture. In addition, we demonstrate the activation of MscL by magnetic field in the presence of the superparamagnetic nanoparticles using patch fluorometry[1]. Materials and Methods: We developed superparamagnetic nanoparticles for activation of the MscL nanovalves by magnetic field. Synthesised CoFe2O4 nanoparticles with the radius less than 10 nm were labelled by SH groups for attachment to MscL. Liposomes made of azolectin (Sigma) were produced using the dehydration/rehydration method as reported previously (Delcour et al. 1989; Häse et al. 1995). Activation of MscL by magnetic field with the nanoparticles attached was examined by the patch clamp technique showing that the number of activated channels under ramp pressure increased upon application of the magnetic field. In addition, we have not observed any cytotoxicity of the nanoparticles in human cultured cells. Nano particles were also characterized by XRD, TEM, FTIR and MPMS. Results and Discussion: By recording the channel activity in patch clamp experiments we found that the number of activated channels increased upon application of the magnetic field in the presence of the nanoparticles, which was not the case in the absence of the magnetic field [Figure 1]. The nanoparticles coated with SH groups on their surface can bind to the cysteine residue of each subunit of the M42C MscL channel mutant by forming a disulphide bond, as previously shown in several EPR and FRET spectroscopic studies. Conclusion: In summary, our study indicates that superparamagnetic nanoparticles of average size of less than 6 nm coated with SH groups are highly suitable as components of a trigger mechanism for opening MscL nanovalves. Due to their very large open pore and property of being modulated by mechanical stimuli MscL channels present an ideal candidate for use as nanovalves in liposomal drug delivery. Consequently, a combination of MscL channels and the magnetic nanoparticles generated for this study holds promise for use in the development of “smart liposomes”, a new generation of liposomal drug delivery system.

Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1–3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4–6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

Study of light-induced MscL gating by EPR spectroscopy.

A number of techniques developed to investigate protein structure and function depend on chemically modifying and/or labeling of proteins. However, in the case of homooligomeric proteins, the presence of multiple identical subunits obstructs the introduction of residue-specific labels to only one or several subunits, selectively. Here, in order to study the initial conformational changes of a homopentameric mechanosensitive ion channel during its gating, we developed a method for labeling a defined number of subunits of the channel with two different cysteine-specific compounds simultaneously. The first one is a light-sensitive channel activator that determines the degree of openness of the ion channel upon irradiation. The second one is a spin label, containing an unpaired electron, which allows following the resulting structural changes upon channel gating by electron paramagnetic resonance spectroscopy. With this method, we could open MscL into different sub-open states. As the number of light switches per channel increased, the intersubunit spin–spin interactions became less, indicating changes in intersubunit proximities and opening of the channel. The ability of controlled activation of MscL into different open states with a noninvasive trigger and following the resulting conformational changes by spectroscopy will pave the way for detailed spectroscopic studies in the area of mechanosensation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-015-1063-4) contains supplementary material, which is available to authorized users.

Magnetic nanoparticles for "smart liposomes".

Liposomal drug delivery systems (LDDSs) are promising tools used for the treatment of diseases where highly toxic pharmacological agents are administered. Currently, destabilising LDDSs by a specific stimulus at a target site remains a major challenge. The bacterial mechanosensitive channel of large conductance (MscL) presents an excellent candidate biomolecule that could be employed as a remotely controlled pore-forming nanovalve for triggered drug release from LDDSs. In this study, we developed superparamagnetic nanoparticles for activation of the MscL nanovalves by magnetic field. Synthesised CoFe2O4 nanoparticles with the radius less than 10 nm were labelled by SH groups for attachment to MscL. Activation of MscL by magnetic field with the nanoparticles attached was examined by the patch clamp technique showing that the number of activated channels under ramp pressure increased upon application of the magnetic field. In addition, we have not observed any cytotoxicity of the nanoparticles in human cultured cells. Our study suggests the possibility of using magnetic nanoparticles as a specific trigger for activation of MscL nanovalves for drug release in LDDSs.

Streptomycin potency is dependent on MscL channel expression.

The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced K+-efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the “hits”. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced K+-efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL’s large pore may provide a mechanism for cell entry.

Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.

Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid–protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.

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 activation mode of the mechanosensitive ion channel, MscL, by lysophosphatidylcholine differs from tension-induced gating.

One of the best-studied mechanosensitive channels is the mechanosensitive channel of large conductance (MscL). MscL senses tension in the membrane evoked by an osmotic down shock and directly couples it to large conformational changes leading to the opening of the channel. Spectroscopic techniques offer unique possibilities to monitor these conformational changes if it were possible to generate tension in the lipid bilayer, the native environment of MscL, during the measurements. To this end, asymmetric insertion of l-α-lysophosphatidylcholine (LPC) into the lipid bilayer has been effective; however, how LPC activates MscL is not fully understood. Here, the effects of LPC on tension-sensitive mutants of a bacterial MscL and on MscL homologs with different tension sensitivities are reported, leading to the conclusion that the mode of action of LPC is different from that of applied tension. Our results imply that LPC shifts the free energy of gating by interfering with MscL-membrane coupling. Furthermore, we demonstrate that the fine-tuned addition of LPC can be used for controlled activation of MscL in spectroscopic studies.

Activation of a Bacterial Mechanosensitive Channel in Mammalian Cells by Cytoskeletal Stress

Cells can sense a myriad of mechanical stimuli. Mechanosensitive channel of large conductance (MscL) found in bacteria is a well-characterized mechanosensitive channel that rapidly responds to an increase in turgor pressure. Functional expression of MscL in mammalian cells has recently been demonstrated, revealing that molecular delivery or transport can be achieved by charge-induced activation of MscL. Despite a well-accepted mechanism for MscL activation by membrane tension in bacteria, it is not clear whether and how MscL can be opened by other modes of force transduction in mammalian cells. In this work, we used a variety of techniques to characterize the gating of MscL expressed in mammalian cells, using both wild type and a G22S mutant which activates at a lower threshold. In particular, employing a new technique, acoustic tweezing cytometry (ATC), we show that ultrasound actuation of integrin-bound microbubbles can lead to MscL opening and that ATC induced MscL activation was dependent on the functional linkage of the microbubbles with an intact actin cytoskeleton. Our results indicate that localized mechanical stress can mediate opening of MscL that requires force transduction through the actin cytoskeleton, revealing a new mode of MscL activation that may prove to be a useful tool for mechanobiology and drug delivery research.

Membrane Properties Affect Opening Behaviors of the Bacterial Mechanosensitive Channel MscL: Molecular Dynamics Study

Mechanosensitive channel MscL is constituted of homopentamer of a subunit with two transmembrane inner (TM1) and outer (TM2) helices and its 3D structure of the closed state has been resolved. TM1s line an ion permeable pore and cross each other near cytoplasmic side, forming the most constricted part of the pore called gate. TM2s face lipids and some amino acids in TM2 act as a tension sensor. MscL is activated by sensing membrane tension and the major issue of MscL is to understand the gating mechanism driven by membrane tension. Previous studies propose that MscL embedded in thin membrane can activate with a lower threshold than that embedded in thick membrane. However, it remains unclear how MscL activation (opening of the gate) depends on membrane thickness in detail. In this study, we performed molecular dynamics (MD) simulations of MscL embedded in four types of lipid bilayer with different membrane thickness (DLPC, DMPC, DPPC and DSPC) to get insight into the dependency at an atomic level. As a result, it was shown also in our results that MscL in a thin lipid bilayer DLPC opened more widely than that embedded in a thick lipid bilayer DSPC. Furthremore, it was found that the thinner membrane tended to make the transmembrane helices of MscL tilt more largely. In order to check MscL-lipid interactions, we calculated the interaction energy between MscL and lipids and found that the interaction energy between Phe93 and lipids, located at the cytoplasmic side, was smaller as the membrane was thinner. This seems to be due to a hydrophobic mismatch between MscL and lipids, which affect the tilting of transmembrane helices followed by expanding of the gate during opening.

Gating Mechanism of Mechanosensitive Ion Channels Studied by Continuum Mechanics

To complement the existing experimental and computational methods used for studies of membrane protein structure and dynamics a finite element (FE) model for multi length-scale and time-scale investigations of the gating mechanism of mechanosensitive (MS) ion channels has been established. The main advantage of the FE simulation over molecular dynamic simulation is its flexibility in handling complex geometries and boundary conditions. In this study the FE model of MscL, the large conductance bacterial MS channel, has been developed as an initial template providing a continuum structural framework for a mechanistic understanding of the gating mechanism in MS channels, which can also be employed to develop structural dynamic models of other types of MS channels. A typical tensional force needed to activate MS channels was first applied to the FE model of the lipid bilayer and was then varied depending on the mechanical properties and cholesterol content of the membrane bilayer surrounding MscL. In agreement with the experimental results the FE model showed that adding cholesterol to the lipid bilayer caused a significant increase in the tensional force required for MscL activation. Furthermore, the current model helped to define the membrane stress distribution around a ‘hole’ in the membrane bilayer containing the MscL protein as well as to identify stress areas where the MscL protein is attached to the hole boundary by applying an appropriate mesh refinement and defining contact conditions between the channel and the bilayer. Cholesterol, although stiffening the lipid bilayer, increased the stress intensity in the highly stressed areas of the MscL protein under tension and thus facilitated rupturing of the lipid bilayer. Although initially surprising, this result is consistent with the patch clamp experiments, which demonstrated that adding cholesterol markedly reduced the bilayer lytic tension.

Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic "emergency release valve," is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E.coli I49 and the equivalent S.aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.