MscS inactivation and recovery are slow voltage-dependent processes sensitive to interactions with lipids.

Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension the channel population readily opens, but under prolonged action of moderate tension it inactivates. The inactivated state is non-conductive and tension insensitive, which suggests that the gate becomes uncoupled from the lipid-facing domains. Because the distinct opening and inactivation transitions are both driven from the closed state by tension transmitted through the lipid bilayer, here we explore how mutations of two conserved positively charged lipid anchors, R46 and R74, affect 1) the rates of opening and inactivation and 2) the voltage dependences of these transitions. Previously estimated kinetic rates for opening-closing transitions in wild-type MscS at low voltages were 3-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that MscS activation exhibits a shallow nearly symmetric dependence on voltage, whereas inactivation is substantially augmented and recovery is slowed down by depolarization. Conversely, hyperpolarization impedes inactivation and speeds up recovery. Mutations of R46 and R74 anchoring the lipid-facing helices to the inner interface to an aromatic residue (W) do not substantially change the activation energy and closing rates, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data clearly delineate the activation-closing and the inactivation-recovery pathways and strongly suggest that only the latter involves extensive rearrangements of the protein-lipid boundary associated with the uncoupling of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery suggests that membrane potential is one of the factors that regulates osmolyte release valves by putting them either on "ready" or "standby" based on the cell's metabolic state.

Dissipation during the Gating Cycle of the Bacterial Mechanosensitive Ion Channel Approaches the Landauer Limit.

The Landauer principle sets a thermodynamic bound of kBT ln 2 on the energetic cost of erasing each bit of information. It holds for any memory device, regardless of its physical implementation. It was recently shown that carefully built artificial devices can attain this bound. In contrast, biological computation-like processes, e.g., DNA replication, transcription and translation use an order of magnitude more than their Landauer minimum. Here, we show that reaching the Landauer bound is nevertheless possible with biological devices. This is achieved using a mechanosensitive channel of small conductance (MscS) from E. coli as a memory bit. MscS is a fast-acting osmolyte release valve adjusting turgor pressure inside the cell. Our patch-clamp experiments and data analysis demonstrate that under a slow switching regime, the heat dissipation in the course of tension-driven gating transitions in MscS closely approaches its Landauer limit. We discuss the biological implications of this physical trait.

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.

Partitioning of Seven Different Classes of Antibiotics into LPS Monolayers Supports Three Different Permeation Mechanisms through the Outer Bacterial Membrane.

The outer membrane (OM) of Gram-negative (G-) bacteria presents a barrier for many classes of antibacterial agents. Lipopolysaccharide (LPS), present in the outer leaflet of the OM, is stabilized by divalent cations and is considered to be the major impediment for antibacterial agent permeation. However, the actual affinities of major antibiotic classes toward LPS have not yet been determined. In the present work, we use Langmuir monolayers formed from E. coli Re and Rd types of LPS to record pressure-area isotherms in the presence of antimicrobial agents. Our observations suggest three general types of interactions. First, some antimicrobials demonstrated no measurable interactions with LPS. This lack of interaction in the case of cefsulodin, a third-generation cephalosporin antibiotic, correlates with its low efficacy against G- bacteria. Ampicillin and ciprofloxacin also show no interactions with LPS, but in contrast to cefsulodin, both exhibit good efficacy against G- bacteria, indicating permeation through common porins. Second, we observe substantial intercalation of the more hydrophobic antibiotics, novobiocin, rifampicin, azithromycin, and telithromycin, into relaxed LPS monolayers. These largely repartition back to the subphase with monolayer compression. We find that the hydrophobic area, charge, and dipole all show correlations with both the mole fraction of antibiotic retained in the monolayer at the monolayer-bilayer equivalence pressure and the efficacies of these antibiotics against G- bacteria. Third, amine-rich gentamicin and the cationic antimicrobial peptides polymyxin B and colistin show no hydrophobic insertion but are instead strongly driven into the polar LPS layer by electrostatic interactions in a pressure-independent manner. Their intercalation stably increases the area per molecule (by up to 20%), which indicates massive formation of defects in the LPS layer. These defects support a self-promoted permeation mechanism of these antibiotics through the OM, which explains the high efficacy and specificity of these antimicrobials against G- bacteria.

Mechanism of Catalysis by l-Asparaginase.

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.

The host-defense peptide piscidin P1 reorganizes lipid domains in membranes and decreases activation energies in mechanosensitive ion channels.

The host-defense peptide (HDP) piscidin 1 (P1), isolated from the mast cells of striped bass, has potent activities against bacteria, viruses, fungi, and cancer cells and can also modulate the activity of membrane receptors. Given its broad pharmacological potential, here we used several approaches to better understand its interactions with multicomponent bilayers representing models of bacterial (phosphatidylethanolamine (PE)/phosphatidylglycerol) and mammalian (phosphatidylcholine/cholesterol (PC/Chol)) membranes. Using solid-state NMR, we solved the structure of P1 bound to PC/Chol and compared it with that of P3, a less potent homolog. The comparison disclosed that although both peptides are interfacially bound and α-helical, they differ in bilayer orientations and depths of insertion, and these differences depend on bilayer composition. Although Chol is thought to make mammalian membranes less susceptible to HDP-mediated destabilization, we found that Chol does not affect the permeabilization effects of P1. X-ray diffraction experiments revealed that both piscidins produce a demixing effect in PC/Chol membranes by increasing the fraction of the Chol-depleted phase. Furthermore, P1 increased the temperature required for the lamellar-to-hexagonal phase transition in PE bilayers, suggesting that it imposes positive membrane curvature. Patch-clamp measurements on the inner Escherichia coli membrane showed that P1 and P3, at concentrations sufficient for antimicrobial activity, substantially decrease the activating tension for bacterial mechanosensitive channels. This indicated that piscidins can cause lipid redistribution and restructuring in the microenvironment near proteins. We conclude that the mechanism of piscidin's antimicrobial activity extends beyond simple membrane destabilization, helping to rationalize its broader spectrum of pharmacological effects.

Recovery of Equilibrium Free Energy from Nonequilibrium Thermodynamics with Mechanosensitive Ion Channels in E. coli.

In situ measurements of the free energy difference between the open and closed states of ion channels are challenging due to hysteresis effects and inactivation. Exploiting recent developments in statistical physics, we present a general formalism to extract the free energy difference ΔF between the closed and open states of mechanosensitive ion channels from nonequilibrium work distributions associated with the opening and closing of the channels (gating) in response to ramp stimulation protocols recorded in native patches. We show that the work distributions obtained from the gating of MscS channels in E. coli membrane satisfy the strong symmetry relation predicted by the Crooks fluctuation theorem. Our approach enables the determination of ΔF using patch-clamp experiments, which are often inherently restricted to the nonequilibrium regime.

Differential Interactions of Piscidins with Phospholipids and Lipopolysaccharides at Membrane Interfaces.

Piscidins 1 and 3 (P1 and P3) are potent antimicrobial peptides isolated from striped bass. Their mechanism of action involves formation of amphipathic α-helices on contact with phospholipids and destabilization of the microbial cytoplasmic membrane. The peptides are active against both Gram-positive and Gram-negative bacteria, suggesting easy passage across the outer membrane. Here, we performed a comparative study of these two piscidins at the air-water interface on lipopolysaccharide (LPS) monolayers modeling the outer bacterial surface of Gram-negative organisms and on phospholipid monolayers, which mimic the inner membrane. The results show that P1 and P3 are highly surface active (log KAW ∼ 6.8) and have similar affinities to phospholipid monolayers (log Klip ≈ 7.7). P1, which is more potent against Gram negatives, exhibits a much stronger partitioning into LPS monolayers (log KLPS = 8.3). Pressure-area isotherms indicate that under increasing lateral pressures, inserted P1 repartitions from phospholipid monolayers back to the subphase or to a more shallow position with in-plane areas of ∼170 Å2 per peptide, corresponding to fully folded amphipathic α-helices. In contrast, peptide expulsion from LPS occurs with areas of ∼35 Å2, suggesting that the peptides may not form the similarly oriented, rigid secondary structures when they avidly intercalate between LPS molecules. Patch-clamp experiments on Escherichia coli spheroplasts show that when P1 and P3 reach the outer surface of the bacterial cytoplasmic membrane, they produce fluctuating conductive structures at voltages above 80 mV. The data suggests that the strong activity of these piscidins against Gram-negative bacteria begins with the preferential accumulation of peptides in the outer LPS layer followed by penetration into the periplasm, where they form stable amphipathic α-helices upon contact with phospholipids and attack the energized inner membrane.

Spatiotemporal relationships defining the adaptive gating of the bacterial mechanosensitive channel MscS.

Adaptive desensitization and inactivation are common properties of most ion channels and receptors. The mechanosensitive channel of small conductance MscS, which serves as a low-threshold osmolyte release valve in most bacteria, inactivates not from the open, but from the resting state under moderate tensions. This mechanism enables the channel to respond differently to slow tension ramps versus abruptly applied stimuli. In this work, we present a reconstruction of the energy landscape for MscS transitions based on patch current kinetics recorded under special pressure protocols. The data are analyzed with a three-state continuous time Markov model, where the tension-dependent transition rates are governed by Arrhenius-type relations. The analysis provides assignments to the intrinsic opening, closing, inactivation, and recovery rates as well as their tension dependencies. These parameters, which define the spatial (areal) distances between the energy wells and the positions of barriers, describe the tension-dependent distribution of the channel population between the three states and predict the experimentally observed dynamic pulse and ramp responses. Our solution also provides an analytic expression for the area of the inactivated state in terms of two experimentally accessible parameters: the tension at which inactivation probability is maximized, γ*, and the midpoint tension for activation, γ0.5. The analysis initially performed on Escherichia coli MscS shows its applicability to the recently characterized MscS homolog from Pseudomonas aeruginosa. Inactivation appears to be a common property of low-threshold MscS channels, which mediate proper termination of the osmotic permeability response and contribute to the environmental fitness of bacteria.

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels.

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.

High-Affinity Interactions of Beryllium(2+) with Phosphatidylserine Result in a Cross-Linking Effect Reducing Surface Recognition of the Lipid.

Beryllium has multiple industrial applications, but its manufacture is associated with a serious occupational risk of developing chronic inflammation in the lungs known as berylliosis, or chronic beryllium disease. Although the Be2+-induced abnormal immune responses have recently been linked to a specific MHC-II allele, the nature of long-lasting granulomas is not fully understood. Here we show that Be2+ binds with a micromolar affinity to phosphatidylserine (PS), the major surface marker of apoptotic cells. Isothermal titration calorimetry indicates that, like that of Ca2+, binding of Be2+ to PS liposomes is largely entropically driven, likely by massive desolvation. Be2+ exerts a compacting effect on PS monolayers, suggesting cross-linking through coordination by both phosphates and carboxyls in multiple configurations, which were visualized in molecular dynamics simulations. Electrostatic modification of PS membranes by Be2+ includes complete neutralization of surface charges at ∼30 µM, accompanied by an increase in the boundary dipole potential. The data suggest that Be2+ can displace Ca2+ from the surface of PS, and being coordinated in a tight shell of four oxygens, it can mask headgroups from Ca2+-mediated recognition by PS receptors. Indeed, 48 µM Be2+ added to IC-21 cultured macrophages specifically suppresses binding and engulfment of PS-coated silica beads or aged erythrocytes. We propose that Be2+ adsorption at the surface of apoptotic cells may potentially prevent normal phagocytosis, thus causing accumulation of secondary necrotic foci and the resulting chronic inflammation.

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells.

L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types. Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers.

Mechanosensitive channels in microbes.

All cells, including microbes, detect and respond to mechanical forces, of which osmotic pressure is most ancient and universal. Channel proteins have evolved such that they can be directly stretched open when the membrane is under turgor pressure. Osmotic downshock, as in rain, opens bacterial mechanosensitive (MS) channels to jettison osmolytes, relieving pressure and preventing cell lysis. The ion flux through individual channel proteins can be observed directly with a patch clamp. MS channels of large and small conductance (MscL and MscS, respectively) have been cloned, crystallized, and subjected to biophysical and genetic analyses in depth. They are now models to scrutinize how membrane forces direct protein conformational changes. Eukaryotic microbes have homologs from animal sensory channels of the TRP superfamily. The MS channel in yeast is also directly sensitive to membrane stretch. This review examines the key concept that proteins embedded in the lipid bilayer can respond to the changes in the mechanical environment the lipid bilayer provides.

Properties of diphytanoyl phospholipids at the air-water interface.

Diphytanoylphosphatidyl choline (DPhPC) is a synthetic ester lipid with methylated tails found in archaeal ether lipids. Because of the stability of DPhPC bilayers and the absence of phase transitions over a broad range of temperatures, the lipid is used as an artificial membrane matrix for the reconstitution of channels, pumps, and membrane-active peptides. We characterized monomolecular films made of DPhPC and its natural ether analog DOPhPC at the air-water interface. We measured compression isotherms and dipole potentials of films made of DPhPC, DPhPE, and DOPhPC. We determined that at 40 mN/m the molecular area of DPhPC is 81.2 Å(2), consistent with X-ray and neutron scattering data obtained in liposomes. This indicates that 40 mN/m is the monolayer-bilayer equivalence pressure for this lipid. At this packing density, the compressibility modulus (Cs(-1 )= 122 ± 7 mN/m) and interfacial dipole potential (V = 355 ± 16 mV) were near their maximums. The molecular dipole moment was estimated to be 0.64 ± 0.02 D. The ether DOPhPC compacted to 70.4 Å(2)/lipid at 40 mN/m displaying a peak compressibility similar to that of DPhPC. The maximal dipole potential of the ether lipid was about half of that for DPhPC at this density, and the elemental dipole moment was about a quarter. The spreading of DPhPC and DOPhPC liposomes reduced the surface tension of the aqueous phase by 46 and 49 mN/m, respectively. This corresponds well to the monolayer collapse pressure. The equilibration time shortened as the temperature increased from 20 to 60 °C, but the surface pressure at equilibrium did not change. The data illustrates the properties of branched chains and the contributions of ester bonds in setting the mechanical and electrostatic parameters of diphytanoyl lipids. These properties determine an environment in which reconstituted voltage- or mechano-activated proteins may function. Electrostatic properties are important in the preparation of asymmetric folded bilayers, whereas lateral compressibility defines the tension in mechanically stimulated droplet interface bilayers.

Membrane Affinity of Platensimycin and Its Dialkylamine Analogs.

Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation.

Molecular force transduction by ion channels: diversity and unifying principles.

Cells perceive force through a variety of molecular sensors, of which the mechanosensitive ion channels are the most efficient and act the fastest. These channels apparently evolved to prevent osmotic lysis of the cell as a result of metabolite accumulation and/or external changes in osmolarity. From this simple beginning, nature developed specific mechanosensitive enzymes that allow us to hear, maintain balance, feel touch and regulate many systemic variables, such as blood pressure. For a channel to be mechanosensitive it needs to respond to mechanical stresses by changing its shape between the closed and open states. In that way, forces within the lipid bilayer or within a protein link can do work on the channel and stabilize its state. Ion channels have the highest turnover rates of all enzymes, and they can act as both sensors and effectors, providing the necessary fluxes to relieve osmotic pressure, shift the membrane potential or initiate chemical signaling. In this Commentary, we focus on the common mechanisms by which mechanical forces and the local environment can regulate membrane protein structure, and more specifically, mechanosensitive ion channels.

On the lipid dependence of bacterial mechanosensitive channel gating in situ.

For bacterial mechanosensitive channels acting as turgor-adjusting osmolyte release valves, membrane tension is the primary stimulus driving opening transitions. Because tension is transmitted through the surrounding lipid bilayer, it is possible that the presence or absence of different lipid species may influence the function of these channels. In this work, we characterize the lipid dependence of chromosome-encoded MscS and MscL in E. coli strains with genetically altered lipid composition. We use two previously generated strains that lack one or two major lipid species (PE, PG, or CL) and engineer a third strain that is highly enriched in CL due to the presence of hyperactive cardiolipin synthase ClsA. We characterize the functional behavior of these channels using patch-clamp and quantify the relative tension midpoints, closing rates, inactivation depth, and the rate of recovery back to the closed state. We also measure the osmotic survival of lipid-deficient strains, which characterizes the functional consequences of lipid-mediated channel function at the cell level. We find that the opening and closing behavior of MscS and MscL tolerate the absence of specific lipid species remarkably well. The lack of cardiolipin (CL), however, reduces the active MscS population relative to MscL and decreases the closing rate, slightly increasing the propensity of MscS toward inactivation and slowing the recovery process. The data points to the robustness of the osmolyte release system and the importance of cardiolipin for the adaptive behavior of MscS.

The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel mechanosensitive channel of small conductance.

Vibrio cholerae adapts to osmotic down-shifts by releasing metabolites through two mechanosensitive (MS) channels, low-threshold MscS and high-threshold MscL. To investigate each channel's contribution to the osmotic response, we generated ΔmscS, ΔmscL, and double ΔmscL ΔmscS mutants in V. cholerae O395. We characterized their tension-dependent activation in patch-clamp, and the millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models describing osmolyte and water fluxes. We illustrate the sequence of events and define the parameters that characterize discrete phases of the osmotic response. Survival is correlated to the extent of cell swelling, the rate of osmolyte release, and the completeness of post-shock membrane resealing. Not only do the two channels interact functionally, but there is also an up-regulation of MscS in the ΔmscL strain, suggesting transcriptional crosstalk. The data reveal the role of MscS in the termination of the osmotic permeability response in V. cholerae.

Polymer-extracted structure of the mechanosensitive channel MscS reveals the role of protein-lipid interactions in the gating cycle.

Membrane protein structure determination is not only technically challenging but is further complicated by the removal or displacement of lipids, which can result in non-native conformations or a strong preference for certain states at the exclusion of others. This is especially applicable to mechanosensitive channels (MSC's) that evolved to gate in response to subtle changes in membrane tension transmitted through the lipid bilayer. E. coli MscS, a model bacterial system, is an ancestral member of the large family of MSCs found across all phyla of walled organisms. As a tension sensor, MscS is very sensitive and highly adaptive; it readily opens under super-threshold tension and closes under no tension, but under lower tensions, it slowly inactivates and can only recover when tension is released. However, existing cryo-EM structures do not explain the entire functional gating cycle of open, closed, and inactivated states. A central question in the field has been the assignment of the frequently observed non-conductive conformation to either a closed or inactivated state. Here, we present a 3 Å MscS structure in native nanodiscs obtained with Glyco-DIBMA polymer extraction, eliminating the lipid removal step that is common to all previous structures. Besides the protein in the non-conductive conformation, we observe well-resolved densities of four endogenous phospholipid molecules intercalating between the lipid-facing and pore-lining helices in preferred orientations. Mutations of positively charged residues coordinating these lipids inhibit MscS inactivation, whereas removal of a negative charge near the lipid-filled crevice increases inactivation. The functional data allows us to assign this class of structures to the inactivated state. This structure reveals preserved lipids in their native locations, and the functional effects of their destabilization illustrate a novel inactivation mechanism based on an uncoupling of the peripheral tension-sensing helices from the gate.