Double Tubular Contractile Structure of the Type VI Secretion System Displays Striking Flexibility and Elasticity.

Antimicrobial treatment can induce many bacterial pathogens to enter a cell wall-deficient state that contributes to persistent infections. The effect of this physiological state on the assembly of transenvelope-anchored organelles is not well understood. The type VI secretion system (T6SS) is a widespread molecular weapon for interspecies interactions and virulence, comprising a long double tubular structure and a transenvelope/baseplate complex. Here, we report that cell wall-deficient spheroplasts assembled highly flexible and elastic T6SS structures forming U, O, or S shapes. Upon contacting the inner membrane, the T6SS tubes did not contract but rather continued to grow along the membrane. Such deformation likely results from continual addition of sheath/tube subunits at the distal end. Induction of TagA repressed curved sheath formation. Curved sheaths could also contract and deliver T6SS substrates and were readily disassembled by the ClpV ATPase after contraction. Our data highlight the dramatic effect of cell wall deficiency on the shape of the T6SS structures and reveal the elastic nature of this double tubular contractile injection nanomachine.IMPORTANCE The cell wall is a physical scaffold that all transenvelope complexes have to cross for assembly. However, the cell wall-deficient state has been described as a common condition found in both Gram-negative and Gram-positive pathogens during persistent infections. Loss of cell wall is known to have pleiotropic physiological effects, but how membrane-anchored large cellular organelles adapt to this unique state is less completely understood. Our study examined the assembly of the T6SS in cell wall-deficient spheroplast cells. We report the elastic nature of contractile T6SS tubules under such conditions, providing key insights for understanding how large intracellular structures such as the T6SS accommodate the multifaceted changes in cell wall-deficient cells.

Sugar enhances outer membrane fusion in Deinococcus grandis spheroplasts to generate calcium ion-dependent extra-huge cells.

In our previous study, we showed that cell fusion occurred in spheroplasts of Deinococcus grandis at 200 mM calcium chloride in the incubation medium. Extra-huge cells (> 0.1 mm in diameter) were observed at this concentration with a low frequency of appearance. In this study, we showed that cell fusion occurred consecutively in D. grandis spheroplasts following an incubation for spheroplast enlargement using medium containing 16.2 mM calcium chloride and 333 mM sucrose. As a result, more extra-huge cells were generated, where cells had maximum diameter of > 1 mm. They can be observed with naked eyes in the incubation medium. The giant cells contained multiple cytoplasms covered by the plasma membrane, indicating that the cell fusion occurred only among the outer membranes. Thus, only the outer membrane and the periplasmic space are shared but not the cytoplasm, indicating that genome of each cell remains in its cytoplasm. Our findings indicate that sugar enhances outer membrane fusion in D. grandis spheroplasts to generate calcium ion-dependent extra-huge cells.

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

Effects of Thermoextracts of Brucella S and L Forms on Lipid Peroxidation and Antioxidant Defense in Organs of Laboratory Animals.

The dynamics of LPO marker malondialdehyde formation and peroxidase-destroying activity was studied in homogenized organs of guinea pigs, immunized with thermoextracts from S and L forms Brucella abortus I-206. The L form brucella thermoextract exhibited a lower reactogenicity and adequately activated the antioxidant system, due to which the destructive effects of ROS could be partially neutralized during the vaccinal process.

Equatorial Assembly of the Cell-Division Actomyosin Ring in the Absence of Cytokinetic Spatial Cues.

The position of the division site dictates the size and fate of daughter cells in many organisms. In animal cells, division-site placement involves overlapping mechanisms, including signaling from the central spindle microtubules, astral microtubules, and spindle poles and through polar contractions [1-3]. In fission yeast, division-site positioning requires overlapping mechanisms involving the anillin-related protein Mid1 and the tip complex (comprising the Kelch-repeat protein Tea1, the Dyrk-kinase Pom1, and the SH3-domain protein Tea4) [4-11]. In addition to these factors, cell shape has also been shown to participate in the maintenance of the position of the actomyosin ring [12-14]. The first principles guiding actomyosin ring placement, however, have not been elucidated in any organism. Because actomyosin ring positioning, ring assembly, and cell morphogenesis are genetically separable in fission yeast, we have used it to derive actomyosin ring placement mechanisms from first principles. We report that, during ring assembly in the absence of cytokinetic cues (anillin-related Mid1 and tip-complex proteins), actin bundles follow the path of least curvature and assemble actomyosin rings in an equatorial position in spherical protoplasts and along the long axis in cylindrical cells and compressed protoplasts. The equatorial position of rings is abolished upon treatment of protoplasts with an actin-severing compound or by slowing down actin polymerization. We propose that the physical properties of actin filaments/bundles play key roles in actomyosin ring assembly and positioning, and that key cytokinetic molecules may modulate the length of actin filaments to promote ring assembly along the short axis.

Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.

PBP1B Glycosyltransferase and Transpeptidase Activities Play Different Essential Roles during the De Novo Regeneration of Rod Morphology in Escherichia coli.

Peptidoglycan is a vital component of nearly all cell wall-bearing bacteria and is a valuable target for antibacterial therapy. However, despite decades of work, there remain important gaps in understanding how this macromolecule is synthesized and molded into a three-dimensional structure that imparts specific morphologies to individual cells. Here, we investigated the particularly enigmatic area of how peptidoglycan is synthesized and shaped during the first stages of creating cell shape de novo, that is, in the absence of a preexisting template. We found that when lysozyme-induced (LI) spheroplasts of Escherichia coli were allowed to resynthesize peptidoglycan, the cells divided first and then elongated to recreate a normal rod-shaped morphology. Penicillin binding protein 1B (PBP1B) was critical for the first stage of this recovery process. PBP1B synthesized peptidoglycan de novo, and this synthesis required that PBP1B interact with the outer membrane lipoprotein LpoB. Surprisingly, when LpoB was localized improperly to the inner membrane, recovering spheroplasts synthesized peptidoglycan and divided but then propagated as amorphous spheroidal cells, suggesting that the regeneration of a normal rod shape depends on a particular spatial interaction. Similarly, spheroplasts carrying a PBP1B variant lacking transpeptidase activity or those in which PBP1A was overproduced could synthesize new peptidoglycan and divide but then grew as oddly shaped spheroids. We conclude that de novo cell wall synthesis requires the glycosyltransferase activity of PBP1B but that PBP1B transpeptidase activity is needed to assemble cell walls with wild-type morphology.IMPORTANCE Bacterial cell wall peptidoglycan is synthesized and modified by penicillin binding proteins (PBPs), which are targeted by about half of all currently prescribed antibiotics, including penicillin and its derivatives. Because antibiotic resistance is rising, it has become increasingly urgent that we fill the gaps in our knowledge about how PBPs create and assemble this protective wall. We report here that PBP1B plays an essential role in synthesizing peptidoglycan in the absence of a preexisting template: its glycosyltransferase activity is responsible for de novo synthesis, while its transpeptidase activity is required to construct cell walls of a specific shape. These results highlight the importance of this enzyme and distinguish its biological roles from those of other PBPs and peptidoglycan synthases.

Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes.

The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.

The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box.

Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a "Jack-In-The-Box" with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure.

Physical properties of Escherichia coli spheroplast membranes.

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.

The mechanoelectrical response of the cytoplasmic membrane of Vibrio cholerae.

Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p0.5MscS/p0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.

The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

Differential effects of lipids and lyso-lipids on the mechanosensitivity of the mechanosensitive channels MscL and MscS.

Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

The desensitization gating of the MthK K+ channel is governed by its cytoplasmic amino terminus.

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance ((1)H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytosolic surface of the plasma membrane.

The fatty acid transport protein (FATP) Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl-coenzyme A synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acids with activation to CoA thioesters. To further define the role of Fat1p in the transport of exogenous fatty acids, the topological orientation of two highly conserved motifs [ATP/AMP and FATP/very long chain acyl CoA synthetase (VLACS)], the carboxyl 124 amino acid residues, which bind the ACSL Faa1p, and the amino and carboxyl termini within the plasma membrane were defined. T7 or hemagglutinin epitope tags were engineered at both amino and carboxyl termini, as well as at multiple nonconserved, predicted random coil segments within the protein. Six different epitope-tagged chimeras of Fat1p were generated and expressed in yeast; the sidedness of the tags was tested using indirect immunofluorescence and protease protection by Western blotting. Plasma membrane localization of the tagged proteins was assessed by immunofluorescence. Fat1p appears to have at least two transmembrane domains resulting in a N(in)-C(in) topology. We propose that Fat1p has a third region, which binds to the membrane and separates the highly conserved residues comprising the two halves of the ATP/AMP motif. The N(in)-C(in) topology results in the placement of the ATP/AMP and FATP/VLACS domains of Fat1p on the inner face of the plasma membrane. The carboxyl-terminal region of Fat1p, which interacts with ACSL, is likewise positioned on the inner face of the plasma membrane. This topological orientation is consistent with the mechanistic roles of both Fat1p and Faa1p or Faa4p in the coupled transport/activation of exogenous fatty acids by vectorial acylation.

Lipid-protein interaction of the MscS mechanosensitive channel examined by scanning mutagenesis.

The mechanosensitive channel of small conductance (MscS) is a bacterial mechanosensitive channel that opens in response to rapid hypoosmotic stress. Since MscS can be opened solely by membrane stretch without help from any accessory protein, the lipid-protein interface must play a crucial role in sensing membrane tension. In this study, the hydrophobic residues in the lipid-protein interface were substituted one by one with a hydrophilic amino acid, asparagine, to modify the interaction between the protein and the lipid. Function of the mutant MscSs was examined by patch-clamp and hypoosmotic shock experiments. An increase in the gating threshold and a decrease in the viability on hypoosmotic shock were observed when the hydrophobic residues near either end of the first or the second transmembrane helix (TM1 or TM2) were replaced with asparagine. This observation indicates that the lipid-protein interaction at the ends of both helices (TM1 and TM2) is essential to MscS function.

Development of a rapid and convenient method for the quantification of HIV-1 budding.

In cells, the expression of Gag protein, one of the major structural proteins of retroviruses, is sufficient for budding virus-like particles (VLPs) from the cell surface. We have previously reported that spheroplasts of Saccharomyces cerevisiae expressing HIV-1 Gag proteins from an episomal plasmid constitutively released a large amount of VLPs into culture media; however, commercially available ELISA kits which detect mature capsid of HIV-1 could not detect uncleaved 55-kDa Gag proteins released from budding yeast. We therefore developed a method to quantitate VLP levels released from budding yeast by using fusion protein from HIV-1 Gag and Firefly Luciferase. This system is useful for screening cellular factor(s) involved in retrovirus budding from S. cerevisiae.

A novel mechanism of autolysis in Helicobacter pylori: possible involvement of peptidergic substances.

BACKGROUND: Helicobacter pylori survival in a hostile acidic environment is known to be caused by its production of urease, which is not released by known secretion pathways. It has been proposed that H. pylori cells undergo spontaneous autolysis during cultivation and that urease becomes surface-associated only concomitant with bacterial autolysis. The aim of this study was to elucidate mechanisms by which H. pylori cells undergo autolysis during cultivation. MATERIALS AND METHODS: Autolysis of H. pylori KZ109 cells was estimated by measuring the turbidity of the culture, by detection of cytoplasmic protein release into the culture supernatant and by scanning electron microscopic observation of H. pylori cells during cultivation. An autolysis-inducing factor (AIF) was partially purified from the culture supernatant by a partition method using ethyl acetate. RESULTS: Bacterial turbidity of KZ109 cells was drastically decreased after late-log phase accompanying release of urease and HspB into the extracellular space. Concomitantly, cell lytic activity was detected in the culture supernatant. Scanning electron microscopic observation suggested that partially purified AIF induced cell lysis. It was also shown that the AIF is different from other autolytic enzymes or substances so far reported. CONCLUSIONS: This study demonstrated the presence of the peptidergic autolytic substances in the culture supernatant of H. pylori KZ109 cells. The results of this study should be useful for further studies aimed at elucidation of the strategy of survival of H. pylori in the gastric environment and elucidation of the mechanisms of pathogenesis induced by H. pylori.

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

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

The "dashpot" mechanism of stretch-dependent gating in MscS.

The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL-, mscS-, mscK-) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose-response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive "dashpot." With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of "dumping" a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information.