Mammalian TRP ion channels are insensitive to membrane stretch.

TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.

Functional similarities between heterogeneously and homogenously expressed MscL constructs.

The mechanosensitive channel of large conductance MscL is a well-characterized mechanically gated non-selective ion channel, which often serves as a prototype mechanosensitive channel for mechanotransduction studies. However, there are some discrepancies between MscL constructs used in these studies, most notably unintended heterogeneous expression from some MscL expression constructs. In this study we investigate the possible cause of this expression pattern, and compare the original non-homogenously expressing constructs with our new homogeneously expressing one to confirm that there is little functional difference between them. In addition, a new MscL construct has been developed with an improved molar extinction coefficient at 280 nm, enabling more accurate protein quantification.

Patch-clamp characterization of the MscS-like mechanosensitive channel from Silicibacter pomeroyi.

Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria.

"Force-From-Lipids" Dependence of the MscCG Mechanosensitive Channel Gating on Anionic Membranes.

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

Flying-patch patch-clamp study of G22E-MscL mutant under high hydrostatic pressure.

High hydrostatic pressure (HHP) present in natural environments impacts on cell membrane biophysical properties and protein quaternary structure. We have investigated the effect of high hydrostatic pressure on G22E-MscL, a spontaneously opening mutant of Escherichia coli MscL, the bacterial mechanosensitive channel of large conductance. Patch-clamp technique combined with a flying-patch device and hydraulic setup allowed the study of the effects of HHP up to 90 MPa (as near the bottom of the Marianas Trench) on the MscL mutant channel reconstituted into liposome membranes, in addition to recording in situ from the mutant channels expressed in E. coli giant spheroplasts. In general, against thermodynamic predictions, hydrostatic pressure in the range of 0.1-90 MPa increased channel open probability by favoring the open state of the channel. Furthermore, hydrostatic pressure affected the channel kinetics, as manifested by the propensity of the channel to gate at subconducting levels with an increase in pressure. We propose that the presence of water molecules around the hydrophobic gate of the G22E MscL channel induce hydration of the hydrophobic lock under HHP causing frequent channel openings and preventing the channel closure in the absence of membrane tension. Furthermore, our study indicates that HHP can be used as a valuable experimental approach toward better understanding of the gating mechanism in complex channels such as MscL.

Bilayer-mediated clustering and functional interaction of MscL channels.

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

Membrane stiffness is one of the key determinants of E. coli MscS channel mechanosensitivity.

Mechanosensitive (MS) channels have an intimate relationship with membrane lipids that underlie their mechanosensitivity. Membrane lipids may influence channel activity by directly interacting with MS channels or by influencing the global properties of the membrane such as elastic area expansion modulus or bending rigidity. Previous work has implicated membrane stiffness as a potential determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing elastic area expansion modulus. Increasing dioleoylphosphatidylethanolamine (DOPE) content of azolectin liposomes made it more difficult to activate EcMscS by membrane tension (i.e. increased gating threshold). This effect was exacerbated by stiffer forms of phosphatidylethanolamine such as the branched chain lipid diphytanoylphosphoethanolamine (DPhPE) or the fully saturated lipid distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Furthermore, a comparison of the branched chain lipid diphytanoylphosphocholine (DPhPC) to the stiffer DPhPE indicated again that it was harder to activate EcMscS in the presence of the stiffer DPhPE. We show that these effects are not due to changes in membrane bending rigidity as the membrane tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 remained the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes caused a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data suggests that membrane stiffness (elastic area expansion modulus) is one of the key determinants of the mechanosensitivity of EcMscS.

Xenon-inhibition of the MscL mechano-sensitive channel and the CopB copper ATPase under different conditions suggests direct effects on these proteins.

Xenon is frequently used as a general anesthetic in humans, but the mechanism remains an issue of debate. While for some membrane proteins, a direct interaction of xenon with the protein has been shown to be the inhibitory mechanism, other membrane protein functions could be affected by changes of membrane properties due to partitioning of the gas into the lipid bilayer. Here, the effect of xenon on a mechanosensitive ion channel and a copper ion-translocating ATPase was compared under different conditions. Xenon inhibited spontaneous gating of the Escherichia coli mechano-sensitive mutant channel MscL-G22E, as shown by patch-clamp recording techniques. Under high hydrostatic pressure, MscL-inhibition was reversed. Similarly, the activity of the Enterococcus hirae CopB copper ATPase, reconstituted into proteoliposomes, was inhibited by xenon. However, the CopB ATPase activity was also inhibited by xenon when CopB was in a solubilized state. These findings suggest that xenon acts by directly interacting with these proteins, rather than via indirect effects by altering membrane properties. Also, inhibition of copper transport may be a novel effect of xenon that contributes to anesthesia.

Tuning ion channel mechanosensitivity by asymmetry of the transbilayer pressure profile.

Mechanical stimuli acting on the cellular membrane are linked to intracellular signaling events and downstream effectors via different mechanoreceptors. Mechanosensitive (MS) ion channels are the fastest known primary mechano-electrical transducers, which convert mechanical stimuli into meaningful intracellular signals on a submillisecond time scale. Much of our understanding of the biophysical principles that underlie and regulate conversion of mechanical force into conformational changes in MS channels comes from studies based on MS channel reconstitution into lipid bilayers. The bilayer reconstitution methods have enabled researchers to investigate the structure-function relationship in MS channels and probe their specific interactions with their membrane lipid environment. This brief review focuses on close interactions between MS channels and the lipid bilayer and emphasizes the central role that the transbilayer pressure profile plays in mechanosensitivity and gating of these fascinating membrane proteins.

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers.

The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.

Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension.

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1-GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels.

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells.

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel.

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a 'virtual sorting' single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations. DOI: http://dx.doi.org/10.7554/eLife.01834.001.

Bacterial mechanosensitive channels: models for studying mechanosensory transduction.

SIGNIFICANCE: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.

Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.

Ion channel studies have been focused on ion channels from animal and human cells over many years. Based on the knowledge acquired, predominantly over the last 20 years, a large diversity of ion channels exists in cellular membranes of prokaryotes as well. Paradoxically, most of what is known about the structure of eukaryotic ion channels is based on the structure of bacterial channels. This is largely due to the suitability of bacterial cells for functional and structural studies of biological macromolecules in a laboratory environment. Development of the "giant spheroplast" preparation from E. coli cells was instrumental for functional studies of ion channels in the bacterial cell membrane. Here we describe detailed protocols used for the preparation of giant spheroplasts as well as protocols used for the patch-clamp recording of native or heterologously expressed ion channels in E. coli spheroplast membrane.

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure.

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the "flying-patch" patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by "shielding" the cytoplasmic domain of the channels.

Studying mechanosensitive ion channels using liposomes.

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.