Chimeras reveal a single lipid-interface residue that controls MscL channel kinetics as well as mechanosensitivity.
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ABSTRACT:
SUBMITTER: Yang LM
PROVIDER: S-EPMC3593973 | biostudies-literature | 2013 Feb
REPOSITORIES: biostudies-literature
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Project description:The bacterial mechanosensitive channel of large conductance (MscL) serves as a biological emergency release valve, preventing the occurrence of cell lysis caused by acute osmotic stress. Its tractable nature allows it to serve as a paradigm for how a protein can directly sense membrane tension. Although much is known of the importance of the hydrophobicity of specific residues in channel gating, it has remained unclear whether electrostatics at the membrane plays any role. We studied MscL chimeras derived from functionally distinct orthologues: Escherichia coli and Staphylococcus aureus. Dissection of one set led to an observation that changing the charge of a single residue, K101, of E. coli (Ec)-MscL, effects a channel phenotype: when mutated to a negative residue, the channel is less mechanosensitive and has longer open dwell times. Assuming electrostatic interactions, we determined whether they are due to protein-protein or protein-lipid interactions by performing site-directed mutagenesis elsewhere in the protein and reconstituting channels into defined lipids, with and without negative head groups. We found that although both interactions appear to play some role, the primary determinant of the channel phenotype seems to be protein-lipid electrostatics. The data suggest a model for the role of electrostatic interactions in the dynamics of MscL gating.
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Project description:MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.
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Project description:The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.
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Project description:YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670-26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19-->Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET(+), thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an approximately 800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19-->Cys MscL in the presence of YggB, but a downshock of only approximately 400 mmol/kg is required in the absence of YggB. In parallel, Leu-19-->Cys MscL, stretched open by large sustained suction in the presence of MTSET(+) in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.
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Project description:Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli.
| S-EPMC5635901 | biostudies-literature
Project description:Temperature-sensitive transient receptor potential vanilloid (thermoTRPV) channels are activated by ligands and heat, and are involved in various physiological processes. ThermoTRPV channels possess a large cytoplasmic ring consisting of N-terminal ankyrin repeat domains (ARD) and C-terminal domains (CTD). The cytoplasmic inter-protomer interface is unique and consists of a CTD coiled around a ?-sheet which makes contacts with the neighboring ARD. Despite much existing evidence that the cytoplasmic ring is important for thermoTRPV function, the mechanism by which this unique structure is involved in thermoTRPV gating has not been clear. Here, we present cryo-EM and electrophysiological studies which demonstrate that TRPV3 gating involves large rearrangements at the cytoplasmic inter-protomer interface and that this motion triggers coupling between cytoplasmic and transmembrane domains, priming the channel for opening. Furthermore, our studies unveil the role of this interface in the distinct biophysical and physiological properties of individual thermoTRPV subtypes.
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Project description:Mechanosensitivity in voltage-gated calcium channels could be an asset to calcium signaling in healthy cells or a liability during trauma. Recombinant N-type channels expressed in HEK cells revealed a spectrum of mechano-responses. When hydrostatic pressure inflated cells under whole-cell clamp, capacitance was unchanged, but peak current reversibly increased ~1.5-fold, correlating with inflation, not applied pressure. Additionally, stretch transiently increased the open-state inactivation rate, irreversibly increased the closed-state inactivation rate, and left-shifted inactivation without affecting the activation curve or rate. Irreversible mechano-responses proved to be mechanically accelerated components of run-down; they were not evident in cell-attached recordings where, however, reversible stretch-induced increases in peak current persisted. T-type channels (alpha(1I) subunit only) were mechano-insensitive when expressed alone or when coexpressed with N-type channels (alpha(1B) and two auxiliary subunits) and costimulated with stretch that augmented N-type current. Along with the cell-attached results, this differential effect indicates that N-type mechanosensitivity did not depend on the recording situation. The insensitivity of T-type currents to stretch suggested that N-type mechano-responses might arise from primary/auxiliary subunit interactions. However, in single-channel recordings, N-type currents exhibited reversible stretch-induced increases in NP(o) whether the alpha(1B) subunit was expressed alone or with auxiliary subunits. These findings set the stage for the molecular dissection of calcium current mechanosensitivity.
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Project description: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.
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Project description:The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.
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