Mechanosensitive Channel Of Small Conductance Research Articles

The MYC transcription factor PbrMYC8 negatively regulates PbrMSL5 expression to promote pollen germination in Pyrus

The successful germination of pollen is essential for double fertilization in flowering plants. Mechanosensitive channels of small conductance (MscS-like, MSL) inhibit pollen germination and maintains cellular integrity of pollen during this process. Therefore, it is vital to carefully regulate the expression of MSL to promote successful pollen germination. Despite its importance, the molecular mechanisms governing MSL expression in plants remain poorly understood. Here, we had identified 15 MSL genes in the pear, among which PbrMSL5 was expressed in pollen development. Subcellular localization experiments revealed that PbrMSL5 was located in both plasma membrane and cytoplasm. Functional investigations, including complementation experiments using the atmsl8 mutant background, demonstrated the involvement of PbrMSL5 in preserving pollen cell integrity and inhibiting germination. Antisense oligonucleotide experiments further confirmed that PbrMSL5 suppressed pear pollen germination by reducing osmotic pressure and Cl− content. Yeast one-hybrid, electrophoretic mobility shift assays, and dual luciferase reporter assay elucidated that PbrMYC8 interacts directly with the N-box element, leading to the suppression of PbrMSL5 expression and promoted pollen germination. These results represented a significant advancement in unraveling the molecular mechanisms controlling plant MSL expression. This study showed valuable contribution to advancing our comprehension of the mechanism underlying pollen germination.

Mechanosensitive Channels: Utilising the Bacterial Cell’s Defence Mechanism against It

Mechanosensitive channels are integral membrane proteins in bacterial cell membranes as well as archaea, and eukaryotes. The role of these bacterial mechanosensitive channels is essential for protecting cells from structural damage during hypoosmotic shock. Mechanosensitive channel of small conductance (MscS) and mechanosensitive channels of large conductance (MscL) are the predominant channels in E. coli. Activation of mechanosensitive channels typically occurs when the bacterial membrane senses tension or distortion. Overall, the ecological niche of bacteria shapes the selective pressures acting on mechanosensitive channels, leading to their adaptation to specific environmental conditions. Bacterial mechanosensitive channels contribute to the ability of bacterial pathogens to adapt to and survive within the host environment, as well as to modulate interactions with host cells and tissues during infection. They could also act as entrance gates for specific antibiotic classes into bacterial cells. Accordingly, it was discovered that nitrofuran nifuroxazide and tuberactinomycin viomycin depended on both Ec-MscS and Ec-MscL for enhanced efficacy. Several compounds have been identified that directly target mechanosensitive channels. For example, ramizol has been shown to reduce the gating threshold of MscL and MscS channels, while styrylbenzene inhibits MscL channels in S. aureus, Streptococcus pneumoniae, and Clostridium difficile. Bacterial mechanosensitive channels are versatile drug targets due to their role in promoting bacterial virulence and host colonization, serving as entry points for antibiotics, and being structurally distinct from mammalian counterparts. These special structures which are meant to protect the cell can also be harnessed as drug targets, thereby increasing the susceptibility of the bacterial cell to target antibiotics. This feature has been harnessed to reduce the burden of antibiotics resistance and its attendant effect on global healthcare complications. The aim of this review is to examine the various physiological, drug resistance and pathogenicity roles of these mechanosensitive channels as well as their usefulness as potential drug targets.

Direct and Continuous Monitoring of Multicomponent Antibiotic Gentamicin in Blood at Single-Molecule Resolution.

Point-of-care monitoring of small molecules in biofluids is crucial for clinical diagnosis and treatment. However, the inherent low degree of recognition of small molecules and the complex composition of biofluids present significant obstacles for current detection technologies. Although nanopore sensing excels in the analysis of small molecules, the direct detection of small molecules in complex biofluids remains a challenge. In this study, we present a method for sensing the small molecule drug gentamicin in whole blood based on the mechanosensitive channel of small conductance in Pseudomonas aeruginosa (PaMscS) nanopore. PaMscS can directly detect gentamicin and distinguish its main components with only a monomethyl difference. The 'molecular sieve' structure of PaMscS enables the direct measurement of gentamicin in human whole blood within 10 min. Furthermore, a continuous monitoring device constructed based on PaMscS achieved continuous monitoring of gentamicin in live rats for approximately 2.5 h without blood consumption, while the drug components can be analyzed in situ. This approach enables rapid and convenient drug monitoring with single-molecule level resolution, which can significantly lower the threshold for drug concentration monitoring and promote more efficient drug use. Moreover, this work also lays the foundation for the future development of continuous monitoring technology with single-molecule level resolution in the living body.

Origin, evolution and diversification of plant mechanosensitive channel of small conductance-like (MSL) proteins

Mechanosensitive (MS) ion channels provide efficient molecular mechanism for transducing mechanical forces into intracellular ion fluxes in all kingdoms of life. The mechanosensitive channel of small conductance (MscS) was one of the best-studied MS channels and its homologs (MSL, MscS-like) were widely distributed in cell-walled organisms. However, the origin, evolution and expansion of MSL proteins in plants are still not clear. Here, we identified more than 2100 MSL proteins from 176 plants and conducted a broad-scale phylogenetic analysis. The phylogenetic tree showed that plant MSL proteins were divided into three groups (I, II and III) prior to the emergence of chlorophytae algae, consistent with their specific subcellular localization. MSL proteins were distributed unevenly into each of plant species, and four parallel expansion was identified in angiosperms. In Brassicaceae, most MSL duplicates were derived by whole-genome duplication (WGD)/segmental duplications. Finally, a hypothetical evolutionary model of MSL proteins in plants was proposed based on phylogeny. Our studies illustrate the evolutionary history of the MSL proteins and provide a guide for future functional diversity analyses of these proteins in plants.

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

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

Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

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.

Mitochondrial function is regulated by mechanosensitive channels in Trypanosoma brucei.

Mechanosensation is an evolutionary conserved trait that allows cells to sense their external and internal conditions, effectively responding to changes in their environment. Beyond the canonical functions such as volume regulation and surface sensing, intracellular mechanosensitive channels play a role in maintaining organelles physiological functions. We have identified a mechanosensitive channel (TbrMscS) homologous to the small conductance mechanosensitive channel found in E. coli. Expression in spheroplasts followed by electrophysiological recordings indicate that TbrMscS is directly activated by tension and has a conductance of 0.2 pS. When expressed in E. coli strains lacking mechanosensitive channels, TbrMscS complemented the function of the mutants, restoring their ability to regulate volume upon osmotic shock. These results confirmed the nature of TbrMscS as a mechanically gated channel and showed the conservation of its role as an osmotic relief valve. In the parasites, the channel is expressed in both life stages, with higher levels in procyclic forms, and localizes to the mitochondria, as expected by its genetic homology with bacterial-like channels. Upon downregulation of the mechanosensitive channel, we observed reduced growth of the parasites, defects in cell volume regulation under hyperosmotic conditions and absence of social motility behavior on semi-solid agar plates. Furthermore, reduction in TbrMscS expression caused dissipation of the mitochondrial membrane potential, showing its participation in the regulation of this organelle. Our results provide the first evidence of mechanosensation in T. brucei and its role in mitochondrial homeostasis in protozoan parasites.

How Functional Lipids Affect the Structure and Gating of Mechanosensitive MscS-like Channels

The ability to cope with and adapt to changes in the environment is essential for all organisms. Osmotic pressure is a universal threat when environmental changes result in an imbalance of osmolytes inside and outside the cell which causes a deviation from the normal turgor. Cells have developed a potent system to deal with this stress in the form of mechanosensitive ion channels. Channel opening releases solutes from the cell and relieves the stress immediately. In bacteria, these channels directly sense the increased membrane tension caused by the enhanced turgor levels upon hypoosmotic shock. The mechanosensitive channel of small conductance, MscS, from Escherichia coli is one of the most extensively studied examples of mechanically stimulated channels. Different conformational states of this channel were obtained in various detergents and membrane mimetics, highlighting an intimate connection between the channel and its lipidic environment. Associated lipids occupy distinct locations and determine the conformational states of MscS. Not all these features are preserved in the larger MscS-like homologues. Recent structures of homologues from bacteria and plants identify common features and differences. This review discusses the current structural and functional models for MscS opening, as well as the influence of certain membrane characteristics on gating.

Structural basis for mechanotransduction in a potassium-dependent mechanosensitive ion channel

Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response to mechanical stress. Amongst these channels, MscK is unique in that its activation also requires external potassium ions. To better understand this dual gating mechanism by force and ligand, we elucidate distinct structures of MscK along the gating cycle using cryo-electron microscopy. The heptameric channel comprises three layers: a cytoplasmic domain, a periplasmic gating ring, and a markedly curved transmembrane domain that flattens and expands upon channel opening, which is accompanied by dilation of the periplasmic ring. Furthermore, our results support a potentially unifying mechanotransduction mechanism in ion channels depicted as flattening and expansion of the transmembrane domain.

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 recently demonstrated that lipid removal by β-cyclodextrin can mimic membrane tension in membrane-scaffold protein-based lipid nanodiscs, providing novel insights into the structural rearrangements that underlie MscS channel gating in response to membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted E.

High-fidelity biosensing of dNTPs and nucleic acids by controllable subnanometer channel PaMscS

Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1–3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.

Self-assembly of polyhedral bilayer vesicles from Piezo ion channels.

Piezo ion channels underlie many forms of mechanosensation in vertebrates and have been found to bend the membrane into strongly curved dome shapes. We develop a methodology describing the self-assembly of lipids and Piezo proteins into polyhedral bilayer vesicles. We validate this methodology for bilayer vesicles formed from bacterial mechanosensitive channels of small conductance, for which experiments found a polyhedral arrangement of proteins with snub cube symmetry and a well-defined characteristic vesicle size. On this basis, we calculate the self-assembly diagram for polyhedral bilayer vesicles formed from Piezo proteins. We find that the radius of curvature of the Piezo dome provides a critical control parameter for the self-assembly of Piezo vesicles, with high abundances of Piezo vesicles with octahedral, icosahedral, and snub cube symmetry with increasing Piezo dome radius of curvature.

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.

Mechanosensitive channel gating by delipidation

The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-β-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.

More Than Just Closed and Open: Unraveling a Mechanosensor

The bacterial mechanosensitive channel of small conductance (MscS) is a well-studied model of how mechanical forces from the membrane can be sensed by an embedded protein. A recent study by Zhang et al. visualizes how MscS behaves under membrane tension, entering a desensitized state when it loses all coordinated lipids.

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.

The Interfacial Electrostatics Defines MscS Gating and Inactivation

The membrane tension that drives transitions in mechanosensitive proteins is transmitted primarily through the polar/apolar interfaces of the lipid bilayer. The mechanosensitive channel of small conductance (MscS) of E. coli has two prominent arginines, R46 and R74, in each subunit which anchor the TM1 and TM2 helices to the cytoplasmic boundary of the membrane. Alignments of 171 MscS homologs show that the presence of positive charges (R, K or H) in these two locations is highly conserved. A distinctive property of WT MscS is that it inactivates under moderate tension, thus properly terminating an osmotic permeability response. Under depolarizing voltages, MscS inactivation is accelerated; hyperpolarization, on the contrary, slows down inactivation and accelerates recovery. Therefore, MscS utilizes normal membrane potential to return back to the resting state. We generated a variety of double mutants at these positions to investigate the interactions with the lipid head groups and examine the voltage dependence of inactivation. We found that N or T substitutions for R46 and R74 produce functional channels, even preserving the voltage sensitivity. The D, A or V mutations produce channels that do not rescue the osmotically sensitive MJF465 strain and do not open in full, demonstrating 10-30% of WT conductance. L, Y and W mutants support the gating and rescue, but remove the voltage dependence of inactivation and recovery. The data indicates that polar interactions between helix-anchoring residues and interfacial lipid phosphates are critical for gating. The transition to the open or inactivated state likely increases the number of favorable contacts with phosphates. Voltage dependence of inactivation can be related to the Lippmann's electrowetting effect, accumulation of co-ions or counter-ions in the double layer on the cytoplasmic surface, which changes the hydration of the TM2-TM3 interface that transmits tension to the gate.

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

Exploring the diversity of mechanosensitive channels in bacterial genomes.

Mechanosensitive ion channels are responsible for touch sensation and proprioception in higher level organisms such as humans and recovery after osmotic stress in bacteria. Bacterial mechanosensitive channels are homologous to either the mechanosensitive channel of large conductance (MscL) or the mechanosensitive channel of small conductance (MscS). In the E. coli genome there are seven unique mechanosensitive channels, a single MscL homologue, and six MscS homologues. The six MscS homologues are members of the diverse MscS superfamily of ion channels, and these channels show variation on both the N and C termini when compared to E. coli MscS. In bacterial strains with phenotypic analysis of the endogenous mechanosensors, the quantity of MscS superfamily members in the genome range from 2 to 6 and all of the strains contain a copy of MscL. Here, we show an in-depth analysis of over 150 diverse bacterial genomes, encompassing nine phyla, to determine the number of genomes that contain an MscL homologue and the average number of MscS superfamily members per genome. We determined that the average genome contains 4 ± 3 MscS homologues and 67% of bacterial genomes encode for a MscL homologue.