Mechanosensitive Channel MscS Research Articles

The dynamic hypoosmotic response of Vibrio cholerae relies on the mechanosensitive channel MscS

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.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

Asymmetric interfacial anchors reveal principles of gating and metabolic control of the bacterial mechanosensitive channel MscS

Asymmetric interfacial anchors reveal principles of gating and metabolic control of the bacterial mechanosensitive channel MscS

In search of a compact closed conformation of bacterial mechanosensitive channel MscS

In search of a compact closed conformation of bacterial mechanosensitive channel MscS

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.

Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response.

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, ∆mscL, ∆mscS, a double knockout ∆mscS ∆mscK, and a triple knockout ∆mscL ∆mscS ∆mscK, in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains, MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the ∆mscL strain, suggesting either a crosstalk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants.

State-specific morphological deformations of the lipid bilayer explain mechanosensitive gating of MSCs ion channels.

The so-called force-from-lipids hypothesis of cellular mechanosensation posits that ion channel gating directly responds to changes in the physical state of the membrane induced for example by lateral tension. Here, we investigate the molecular basis for this transduction mechanism by studying the mechanosensitive ion channel MscS from Escherichia coli and its eukaryotic homolog, MSL1 from Arabidopsis thaliana. First, we use single-particle cryo-EM to determine the structure of a novel open conformation of wild-type MscS, stabilize in thinned lipid nanodisc. Compared with the closed state, the structure shows a reconfiguration of helices TM1, TM2 and TM3a, resulting in widening of the central pore. Based on these structures, we examined how the morphology of the lipid bilayer is altered upon gating, using molecular dynamics simulations. The simulations reveal that, in the absence of extrinsic forces, closed-state MscS causes drastic protrusions in the inner leaflet of the lipid bilayer. These deformations develop to provide adequate solvation to hydrophobic features of the protein surface, and clearly reflect a high energy conformation for the membrane. Strikingly, the protrusions are largely eradicated upon channel opening. An analogous computational study of open and closed states of MSL1 recapitulates these findings. The gating equilibrium of MscS channels thus appears to be dictated by two opposing conformational preferences, namely those of the lipid membrane and of the protein structure. We theorize that membrane tension controls this balance because it increases the energetic cost of the membrane deformations that specifically stabilize the closed channel, shifting the gating equilibrium towards the conductive form.

The role of protein-membrane electrostatic interactions in the adaptive gating cycle of MscS.

Bacterial mechanosensitive channels MscS and MscL directly respond to tension in the cytoplasmic membrane. It appears not to be a coincidence that both channels are anchored to membrane lipids with positively charged residues primarily at the cytoplasmic interface of the inner membrane, leaving the periplasmic side of the barrel the freedom to move and tilt. The functional cycle of MscS involving opening ↔ closing and inactivation ↔ recovery transitions relies on two prominent anchors, R46 and R74, that reside at the level of cytoplasmic phosphates. WT MscS exhibits voltage-dependent inactivation and recovery, showing deeper inactivation and slower recovery under depolarizing voltages (+60 mV) compared to hyperpolarizations (−60 mV). Substitutions of both R46 and R74 with poor interfacial anchors (A or D) result in no expression, whereas G, I, L, or V substitutions express but produce non-functional channels. Patch-clamp experiments showed that polar substitutions in positions 46 and 74, which maintain H-bonding capability to lipid phosphate groups (N, Q, S), preserve expression, activation, voltage-dependence of inactivation and recovery, as well as osmotic rescuing. Aromatic (W, Y) substitutions produce channels that completely lack voltage sensitivity of inactivation and recovery, but are otherwise functional. Insertion of periplasmic anchors (R or W) in flexible N-terminal segments (positions 3-21) did not produce strong phenotypes, however, the A22R substitution, which faces outer-leaflet phosphates, abolishes inactivation without affecting opening. These data show that the inactivation and recovery transitions are sensitive to the polarity of the cytoplasmic ends of TM1 and TM2 and are likely associated with the change of hydration near these segments which can be altered by voltage. The insertion of a periplasmic anchor (R22) restrains the position of the TM1-TM2 pair of helices, which require a higher degree of freedom during inactivation.

Quantifying pressure and ultrasound activation of the mechanosensitive K+ channel TRAAK.

The study of mechanosensitive ion channels depends upon electrophysiological recordings coupled with mechanical stimulation by cell poking, pillar arrays, or pressure application via to excised membrane patches. In each of these environments, mechanical energy is deposited into the membrane and causes the membrane to stretch, inducing tension. Unlike other assays, pressure stimulation with simultaneous imaging of patched membranes permits tension, the relevant gating stimulus for most mechanosensitive channels, to be calculated. Here, we quantify the mechanical tuning of the mechanosensitive K+ channel TRAAK and compare it to the bacterial mechanosensitive channel MscS. We find TRAAK is activated at lower tension values and over a wider range of tension compared to MscS. We further show that ultrasound activates TRAAK and MscS in excised patches. Changes in membrane curvature induced by ultrasound or pressure stimulation resulted in the same degree of channel activation. This suggests ultrasound activation of mechanosensitive ion channels is due to increased membrane tension rather than alternative mechanisms posited for transfer of acoustic energy to channels.

State-specific morphological deformations of the lipid bilayer explain mechanosensitive gating of MscS ion channels.

The force-from-lipids hypothesis of cellular mechanosensation posits that membrane channels open and close in response to changes in the physical state of the lipid bilayer, induced for example by lateral tension. Here, we investigate the molecular basis for this transduction mechanism by studying the mechanosensitive ion channel MscS from Escherichia coli and its eukaryotic homolog MSL1 from Arabidopsis thaliana. First, we use single-particle cryo-electron microscopy to determine the structure of a novel open conformation of wild-type MscS, stabilized in a thinned lipid nanodisc. Compared with the closed state, the structure shows a reconfiguration of helices TM1, TM2, and TM3a, and widening of the central pore. Based on these structures, we examined how the morphology of the membrane is altered upon gating, using molecular dynamics simulations. The simulations reveal that closed-state MscS causes drastic protrusions in the inner leaflet of the lipid bilayer, both in the absence and presence of lateral tension, and for different lipid compositions. These deformations arise to provide adequate solvation to hydrophobic crevices under the TM1-TM2 hairpin, and clearly reflect a high-energy conformation for the membrane, particularly under tension. Strikingly, these protrusions are largely eradicated upon channel opening. An analogous computational study of open and closed MSL1 recapitulates these findings. The gating equilibrium of MscS channels thus appears to be dictated by opposing conformational preferences, namely those of the lipid membrane and of the protein structure. We propose a membrane deformation model of mechanosensation, which posits that tension shifts the gating equilibrium towards the conductive state not because it alters the mode in which channel and lipids interact, but because it increases the energetic cost of the morphological perturbations in the membrane required by the closed state.

The selectivity of the bacterial mechanosensitive channel MscS is tuned to support fast electroneutral osmolyte transport

All free-living microorganisms are subjected to drastic changes in osmolarity. During a sudden osmotic down-shock, bacteria evade lysis by massively ejecting small metabolites through mechanosensitive channels MscS and MscL. Therefore, these channels should select for dispensable intracellular osmolytes. Although the larger MscL is non-selective, the smaller MscS is slightly anionic with a permeability ratio PK+/PCl- of 2:3. When chloride, which is not the dominant intracellular anion, is substituted for acetate, MscS becomes completely non-selective (1:1), strongly suggesting that it is designed to pass carboxylic substances with counterions.

The role of electrostatic interactions in the stabilization of main functional states of the bacterial mechanosensitive channel MscS

Conformational searches initiated from known crystal and cryo-EM structures have suggested a compact resting state of MscS characterized by “upright” TM1-TM2 pairs and tighter TM2-TM3 contact. Simulated transitions from structures with splayed TM1-TM2 pairs to this compact state point to the importance of several conserved arginine residues. Here, we determine the state-stabilizing contributions of these arginines and evaluate the compact model. We found that R46, R54, and R74 likely stabilize the open state by acting as anchors for TM1-TM2 helices to phospholipids of the inner leaflet.

Channels under tension.

This study provides evidence that membrane lipids are important in regulating the dynamics of the bacterial mechanosensitive channel MscS in response to changes in membrane tension.

Visualization of the mechanosensitive ion channel MscS under membrane tension.

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.

Molecular basis of force-from-lipids gating in the mechanosensitive channel MscS.

Prokaryotic mechanosensitive (MS) channels open by sensing the physical state of the membrane. As such, lipid-protein interactions represent the defining molecular process underlying mechanotransduction. Here, we describe cryo-electron microscopy (cryo-EM) structures of the E. coli small-conductance mechanosensitive channel (MscS) in nanodiscs (ND). They reveal a novel membrane-anchoring fold that plays a significant role in channel activation and establish a new location for the lipid bilayer, shifted ~14 Å from previous consensus placements. Two types of lipid densities are explicitly observed. A phospholipid that 'hooks' the top of each TM2-TM3 hairpin and likely plays a role in force sensing, and a bundle of acyl chains occluding the permeation path above the L105 cuff. These observations reshape our understanding of force-from-lipids gating in MscS and highlight the key role of allosteric interactions between TM segments and phospholipids bound to key dynamic components of the channel.

Structure of the Mechanosensitive Channel MscS Embedded in the Membrane Bilayer

Since life has emerged, gradients of osmolytes over the cell membrane cause pressure changes in the cell and require tight regulation to prevent cell rupture. The mechanosensitive channel of small conductance (MscS) releases solutes and water when a hypo-osmotic shock raises the pressure in the cell. It is a member of a large family of MscS-like channels found in bacteria, archaea, fungi and plants and model for mechanosensation. MscS senses the increase of tension in the membrane directly by the force from the lipids, but the molecular mechanism is still elusive. We determined the lipid interactions of MscS by resolving the structure of Escherichia coli MscS embedded in membrane discs to 2.9-Å resolution using cryo-electron microscopy. The membrane is attached only to parts of the sensor paddles of MscS, but phospholipid molecules move through grooves into remote pockets on the cytosolic side. On the periplasmic side, a lipid bound by R88 at the pore entrance is separated from the membrane by TM1 helices. The N-terminus interacts with the periplasmic membrane surface. We demonstrate that the unique membrane domain of MscS promotes deep penetration of lipid molecules and shows multimodal interaction with the membrane to fine-tune tension sensing.

Transmembrane TM3b of Mechanosensitive Channel MscS Interacts With Cytoplasmic Domain Cyto-Helix.

The mechanosensitive channel MscS functions as an osmolyte emergency release-valve in the event of a sudden decrease in external environmental osmolarity. MscS has served as a paradigm for studying how channel proteins detect and respond to mechanical stimuli. However, the inter-domain interactions and structural rearrangements that occur in the MscS gating process remain largely unknown. Here, we determined the interactions between the transmembrane domain and cytoplasmic domain of MscS. Using in vivo cellular viability, single-channel electrophysiological recording, and cysteine disulfide trapping, we demonstrated that N117 of the TM3b helix and N167 of the Cyto-helix are critical residues that function at the TM3b-Cyto helix interface. In vivo downshock assays showed that double cysteine substitution at N117 and N167 failed to rescue the osmotic-lysis phenotype of cells in acute osmotic downshock. Single-channel recordings demonstrated that cysteine cross-linking of N117C and N167C led to a non-conductive channel. Consistently, coordination of the histidines of N117H and N167H caused a decrease in channel gating. Moreover, cross-linked N117 and N167 altered the gating of the severe gain-of-function mutant L109S. Our results demonstrate that N117–N167 interactions stabilize the inactivation state by an association of TM3b segments with β-domains of the cytoplasmic region, providing further insights into the gating mechanism of the MscS channel.

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.

Spatiotemporal Relationships for the Three-State Mechanosensitive Channel MscS

Spatiotemporal Relationships for the Three-State Mechanosensitive Channel MscS

The role of lipids in mechanosensation.

The ability of proteins to sense membrane tension is pervasive in biology. A higher resolution structure of E. coli MscS, the channel of small conductance, identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chain within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (two acyl chains) from MscS pockets and trigger channel opening. We propose the extent of acyl chain interdigitation in these pockets determines the conformation of MscS. Where interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable and the channel gates.