Mechanosensitive Channel Of Large Conductance Research Articles

Selective correlations between aliphatic 13C nuclei in protein solid-state NMR

Solid-state nuclear magnetic resonance (NMR) is a potent tool for studying the structures and dynamics of insoluble proteins. It starts with signal assignment through multi-dimensional correlation experiments, where the aliphatic 13Cα-13Cβ correlation is indispensable for identifying specific residues. However, developing efficient methods for achieving this correlation is a challenge in solid-state NMR. We present a simple band-selective zero-quantum (ZQ) recoupling method, named POST-C4161 (PC4), which enhances 13Cα-13Cβ correlations under moderate magic-angle spinning (MAS) conditions. PC4 requires minimal 13C radio-frequency (RF) field and proton decoupling, exhibits high stability against RF variations, and achieves superior efficiency. Comparative tests on various samples, including the formyl-Met-Leu-Phe (fMLF) tripeptide, microcrystalline β1 immunoglobulin binding domain of protein G (GB1), and membrane protein of mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL), demonstrate that PC4 selectively enhances 13Cα-13Cβ correlations by up to 50 % while suppressing unwanted correlations, as compared to the popular dipolar-assisted rotational resonance (DARR). It has addressed the long-standing need for selective 13C–13C correlation methods. We anticipate that this simple but efficient PC4 method will have immediate applications in structural biology by solid-state NMR.

Blue light-mediated gene expression as a promising strategy to reduce antibiotic resistance in Escherichia coli.

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of β-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.

Molecular Transportation Conversion of Membrane Tension Using a Mechanosensitive Channel in Asymmetric Lipid-Protein Vesicles.

Giant lipid vesicles composed of a lipid bilayer form complex membrane structures and enzyme network reactions that can be used to construct well-defined artificial cell models based on microfluidic technologies and synthetic biology. As a different approach to cell-mimicking systems, we formed an asymmetric lipid-amphiphilic protein (oleosin) vesicle containing a lipid and an oleosin monolayer in the outer and inner leaflets, respectively. These asymmetric vesicles enabled the reconstitution and function of β-barrel types of membrane proteins (OmpG) and the fission of vesicles stimulated by lysophospholipids. These applications combine the advantages of the high stability of lipids and oleosin leaflets in asymmetric lipid-oleosin vesicles. In this study, to evaluate the versatility of this asymmetric lipid-oleosin vesicle, the molecular transport of the mechanosensitive channel of large conductance (MscL) with an α-helix was evaluated by changing the tension of the asymmetric vesicle membrane with lysophospholipid. A nanopore of MscL assembled as a pentamer of MscLs transports small molecules of less than 10 kDa by sensing physical stress at the lipid bilayer. The amount and maximum size of the small molecules transported via MscL in the asymmetric lipid-oleosin vesicles were compared to those in the lipid vesicles. We revealed the existence of the C- and N-terminal regions (cytoplasmic side) of MscL on the inner leaflet of the asymmetric lipid-oleosin vesicles using an insertion direction assay. Furthermore, the change in the tension of the lipid-oleosin membrane activated the proteins in these vesicles, inducing their transportation through MscL nanopores. Therefore, asymmetric lipid-oleosin vesicles containing MscL can be used as substrates to study the external environment response of complex artificial cell models.

Response of heterologously expressed pressure sensor-actuator-modulator macromolecule to external mechanical stress

Cells from different organs in the body experience a range of mechanical and osmotic pressures that change in various diseases, including neurological, cardiovascular, ophthalmological, and renal diseases. Here, we demonstrate the use of an engineered Sensor-Actuator-Modulator (SAM) of microbial origin derived from a mechanosensitive channel of large conductance (MscL) for sensing external mechanical stress and modulating activities of mammalian cells. SAM is reliably expressed in the mammalian cell membrane and acts as a tension-activated pressure release valve. Further, the activities of heterologously expressed SAM in mammalian cells could be modulated by osmotic pressure. A comparison of the mechanosensitive activities of SAM-variants from different microbial origins shows differential inward current and dye uptake in response to mechanical stress exerted by hypo-osmotic shock. The use of SAM channels as mechanical stress-activated modulators in mammalian cells could provide new therapeutic approaches for treating disorders related to mechanical or osmotic pressure.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Mechanosensitive channel of large conductance enhances the mechanical stretching-induced upregulation of glycolysis and oxidative metabolism in Schwann cells

BackgroundPhysical exercise directly stretching the peripheral nerve promotes nerve regeneration; however, its action mechanism remains elusive. Our present study aimed to investigate the effects of mechanosensitive channel of large conductance (MscL) activated by mechanical stretching on the cultured Schwann cells (SCs) and explore the possible mechanism.MethodsPrimary SCs from neonatal mice at 3–5 days of age were derived and transfected with the lentivirus vector expressing a mutant version of MscL, MscL-G22S. We first detected the cell viability and calcium ion (Ca2+) influx in the MscL-G22S-expressing SCs with low-intensity mechanical stretching and the controls. Proteomic and energy metabolomics analyses were performed to investigate the comprehensive effects of MscL-G22S activation on SCs. Measurement of glycolysis- and oxidative phosphorylation-related molecules and ATP production were respectively performed to further validate the effects of MscL-G22S activation on SCs. Finally, the roles of phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in the mechanism of energy metabolism modulation of SCs by MscL-G22S activation was investigated.ResultsMechanical stretching-induced MscL-G22S activation significantly increased the cell viability and Ca2+ influx into the SCs. Both the proteomic and targeted energy metabolomics analysis indicated the upregulation of energy metabolism as the main action mechanism of MscL-G22S-activation on SCs. MscL-G22S-activated SCs showed significant upregulation of glycolysis and oxidative phosphorylation when SCs with stretching alone had only mild upregulation of energy metabolism than those without stimuli. MscL-G22S activation caused significant phosphorylation of the PI3K/AKT/mTOR signaling pathway and upregulation of HIF-1α/c-Myc. Inhibition of PI3K abolished the MscL-G22S activation-induced upregulation of HIF-1α/c-Myc signaling in SCs and reduced the levels of glycolysis- and oxidative phosphorylation-related substrates and mitochondrial activity.ConclusionMechanical stretching activates MscL-G22S to significantly promote the energy metabolism of SCs and the production of energic substrates, which may be applied to enhance nerve regeneration via the glia-axonal metabolic coupling.

Membrane Protein Structures in Native Cellular Membranes Revealed by Solid-State NMR Spectroscopy.

The structural characterization of membrane proteins within the cellular membrane environment is critical for understanding the molecular mechanism in their native functional context. However, conducting residue site-specific structural analysis of membrane proteins in native membranes by solid-state NMR faces challenges due to poor spectral sensitivity and serious interference from background protein signals. In this study, we present a new protocol that combines various strategies for cellular membrane sample preparations, enabling us to reveal the secondary structure of the mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL) in Escherichia coli inner membranes. Our findings demonstrate the feasibility of achieving complete resonance assignments and the potential for determining the 3D structures of membrane proteins within cellular membranes. We find that the use of the BL21(DE3) strain in this protocol is crucial for effectively suppressing background protein labeling without compromising the sensitivity of the target protein. Furthermore, our data reveal that the structures of different proteins exhibit varying degrees of sensitivity to the membrane environment. These results underscore the significance of studying membrane proteins within their native cellular membranes when performing structural characterizations. Overall, this study opens up a new avenue for achieving the atomic-resolution structural characterization of membrane proteins within their native cellular membranes, providing valuable insights into the nativeness of membrane proteins.

Modulation of deep neural circuits with sonogenetics

Noninvasive control of neuronal activity in the deep brain can be illuminating for probing brain function and treating dysfunctions. Here, we present a sonogenetic approach for controlling distinct mouse behavior with circuit specificity and subsecond temporal resolution. Targeted neurons in subcortical regions were made to express a mutant large conductance mechanosensitive ion channel (MscL-G22S), enabling ultrasound to trigger activity in MscL-expressing neurons in the dorsal striatum and increase locomotion in freely moving mice. Ultrasound stimulation of MscL-expressing neurons in the ventral tegmental area could activate the mesolimbic pathway to trigger dopamine release in the nucleus accumbens and modulate appetitive conditioning. Moreover, sonogenetic stimulation of the subthalamic nuclei of Parkinson's disease model mice improved their motor coordination and mobile time. Neuronal responses to ultrasound pulse trains were rapid, reversible, and repeatable. We also confirmed that the MscL-G22S mutant is more effective to sensitize neurons to ultrasound compared to the wild-type MscL. Altogether, we lay out a sonogenetic approach which can selectively manipulate targeted cells to activate defined neural pathways, affect specific behaviors, and relieve symptoms of neurodegenerative disease.

Engineering osmolysis susceptibility in Cupriavidus necator and Escherichia coli for recovery of intracellular products

BackgroundIntracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. ResultsTo overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed.ConclusionsOur strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

ArfA antisense RNA regulates MscL excretory activity.

Excretion of cytoplasmic protein (ECP) is a commonly observed phenomenon in bacteria, and this partial extracellular localisation of the intracellular proteome has been implicated in a variety of stress response mechanisms. In response to hypoosmotic shock and ribosome stalling in Escherichia coli, ECP is dependent upon the presence of the large-conductance mechanosensitive channel and the alternative ribosome-rescue factor A gene products. However, it is not known if a mechanistic link exists between the corresponding genes and the respective stress response pathways. Here, we report that the corresponding mscL and arfA genes are commonly co-located on the genomes of Gammaproteobacteria and display overlap in their respective 3' UTR and 3' CDS. We show this unusual genomic arrangement permits an antisense RNA-mediated regulatory control between mscL and arfA, and this modulates MscL excretory activity in E. coli These findings highlight a mechanistic link between osmotic, translational stress responses and ECP in E. coli, further elucidating the previously unknown regulatory function of arfA sRNA.

Mechanosensitive channel MscL induces non-apoptotic cell death and its suppression of tumor growth by ultrasound.

Mechanosensitive channel of large conductance (MscL) is the most thoroughly studied mechanosensitive channel in prokaryotes. Owing to its small molecular weight, clear mechanical gating mechanism, and nanopore forming ability upon opening, accumulating studies are implemented in regulating cell function by activating mechanosensitive channel of large conductance in mammalian cells. This study aimed to investigate the potentials of mechanosensitive channel of large conductance as a nanomedicine and a mechano-inducer in non-small cell lung cancer (NSCLC) A549 cells from the view of molecular pathways and acoustics. The stable cytoplasmic vacuolization model about NSCLC A549 cells was established via the targeted expression of modified mechanosensitive channel of large conductance channels in different subcellular organelles. Subsequent morphological changes in cellular component and expression levels of cell death markers are analyzed by confocal imaging and western blots. The permeability of mitochondrial inner membrane (MIM) exhibited a vital role in cytoplasmic vacuolization formation. Furthermore, mechanosensitive channel of large conductance channel can be activated by low intensity focused ultrasound (LIFU) in A549 cells, and the suppression of A549 tumors in vivo was achieved by LIFU with sound pressure as low as 0.053MPa. These findings provide insights into the mechanisms underlying non-apoptotic cell death, and validate the nanochannel-based non-invasive ultrasonic strategy for cancer therapy.

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

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

Cell-Free Synthesis Goes Electric: Dual Optical and Electronic Biosensor via Direct Channel Integration into a Supported Membrane Electrode.

Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.

Chanalyzer: A Computational Geometry Approach for the Analysis of Protein Channel Shape and Dynamics.

Morphological analysis of protein channels is a key step for a thorough understanding of their biological function and mechanism. In this respect, molecular dynamics (MD) is a very powerful tool, enabling the description of relevant biological events at the atomic level, which might elude experimental observations, and pointing to the molecular determinants thereof. In this work, we present a computational geometry-based approach for the characterization of the shape and dynamics of biological ion channels or pores to be used in combination with MD trajectories. This technique relies on the earliest works of Edelsbrunner and on the NanoShaper software, which makes use of the alpha shape theory to build the solvent-excluded surface of a molecular system in an aqueous solution. In this framework, a channel can be simply defined as a cavity with two entrances on the opposite sides of a molecule. Morphological characterization, which includes identification of the main axis, the corresponding local radius, and the detailed description of the global shape of the cavity, is integrated with a physico-chemical description of the surface facing the pore lumen. Remarkably, the possible existence or temporary appearance of fenestrations from the channel interior towards the outer lipid matrix is also accounted for. As a test case, we applied the present approach to the analysis of an engineered protein channel, the mechanosensitive channel of large conductance.

Activation of a Bacterial Mechanosensitive Channel, MscL, Underlies the Membrane Permeabilization of Dual-Targeting Antibacterial Compounds

Resistance to antibiotics is a serious and worsening threat to human health worldwide, and there is an urgent need to develop new antibiotics that can avert it. One possible solution is the development of compounds that possess multiple modes of action, requiring at least two mutations to acquire resistance. Compound SCH-79797 both avoids resistance and has two mechanisms of action: one inhibiting the folate pathway, and a second described as “membrane permeabilization”; however, the mechanism by which membranes from bacterial cells, but not the host, are disrupted has remained mysterious. The opening of the bacterial mechanosensitive channel of large conductance, MscL, which ordinarily serves the physiological role of osmotic emergency release valves gated by hypoosmotic shock, has been previously demonstrated to affect bacterial membrane permeabilization. MscL allows the rapid permeabilization of both ions and solutes through the opening of the largest known gated pore, which has a diameter of 30 Å. We found that SCH-79797 and IRS-16, a more potent derivative, directly bind to the MscL channel and produce membrane permeabilization as a result of its activation. These findings suggest that possessing or adding an MscL-activating component to an antibiotic compound could help to lower toxicity and evade antibiotic resistance.

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials.

Decisive structural elements in water and ion permeation through mechanosensitive channels of large conductance: insights from molecular dynamics simulation.

In this paper, a series of equilibrium molecular dynamics simulations (EMD), steered molecular dynamics (SMD), and computational electrophysiology methods are carried out to explore water and ion permeation through mechanosensitive channels of large conductance (MscL). This research aims to identify the pore-lining side chains of the channel in different conformations of MscL homologs by analyzing the pore size. The distribution of permeating water dipole angles through the pore domains enclosed by VAL21 and GLU104 demonstrated that water molecules are oriented toward the charged oxygen headgroups of GLU104 from their hydrogen atoms to retain this interaction in a stabilized fashion. Although, this behavior was not perceived for VAL21. Numerical assessments of the secondary structure clarified that, during the ion permeation, in addition to the secondary structure alterations, the structure of Tb-MscL would also undergo significant conformational changes. It was elucidated that VAL21, GLU104, and water molecules accomplish a fundamental task in ion permeation. The mentioned residues hinder ion permeation so that the pulling SMD force is increased remarkably when the ions permeate through the domains enclosed by VAL21 and GLU102. The hydration level and potassium diffusivity in the hydrophobic gate of the transmembrane domain were promoted by applying the external electric field. Furthermore, the implementation of an external electric field altered the distribution pattern for potassium ions in the system while intensifying the accumulation of Cl− in the vicinity of ARG11 and ARG98.

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

Mechanosensitive channel of large conductance (MscL) detects and responds to changes in the pressure profile of cellular membranes and transduces the mechanical energy into electrical and/or chemical signals. MscL can be activated using ultrasonic or chemical activation methods to improve the absorption of medicines and bioactive compounds into cells. However, re-engineering chemical signals such as pH change can trigger channel activation in MscL. This study elucidates the activation mechanism of an engineered MscL at an atomic level through a combination of equilibrium and non-equilibrium (NE) molecular dynamics (MD) simulations. Comparing the wild-type (WT) and engineered MscLactivation processes suggests that the two systems are likely associated with different active states and different transition pathways. These findings indicate that (1) periplasmic loops play a key role in the activation process of MscL, (2) the loss of various backbone-backbone hydrogen bonds and salt bridge interactions in the engineered MscLchannel causes the spontaneous opening of the channel, and (3) the most significant interactions lost during the activation process are between the transmembrane helices 1 and 2 in engineered MscLchannel. The orientation-based biasing approach for producing and optimizing an open MscL model used in this work is a promising way to characterize unknown protein functional states and investigate the activation processes in ion channels and transmembrane proteins in general. This work paves the way for a computational framework for engineering more efficient pH-sensing mechanosensitive channels.

Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties.

Curcumin, a natural compound isolated from the rhizome of turmeric, has been shown to have antibacterial properties. It has several physiological effects on bacteria including an apoptosis-like response involving RecA, membrane permeabilization, inhibiting septation, and it can also work synergistically with other antibiotics. The mechanism by which curcumin permeabilizes the bacterial membrane has been unclear. Most bacterial species contain a Mechanosensitive channel of large conductance, MscL, which serves the function of a biological emergency release valve; these large-pore channels open in response to membrane tension from osmotic shifts and, to avoid cell lysis, allow the release of solutes from the cytoplasm. Here we show that the MscL channel underlies the membrane permeabilization by curcumin as well as its synergistic properties with other antibiotics, by allowing access of antibiotics to the cytoplasm; MscL also appears to have an inhibitory role in septation, which is enhanced when activated by curcumin.

Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel's sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the "force-from-lipids" principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.