The simplest cell-like structure, the lipid bilayer vesicle, can respond to mechanical deformation by elastic membrane dilation/thinning and curvature changes. When a protein is inserted in the lipid bilayer, an energetic cost may arise because of hydrophobic mismatch between the protein and bilayer. Localized changes in bilayer thickness and curvature may compensate for this mismatch. The peptides alamethicin and gramicidin and the bacterial membrane protein MscL form mechanically gated (MG) channels when inserted in lipid bilayers. Their mechanosensitivity may arise because channel opening is associated with a change in the protein's membrane-occupied area, its hydrophobic mismatch with the bilayer, excluded water volume, or a combination of these effects. As a consequence, bilayer dilation/thinning or changes in local membrane curvature may shift the equilibrium between channel conformations. Recent evidence indicates that MG channels in specific animal cell types (e.g., Xenopus oocytes) are also gated directly by bilayer tension. However, animal cells lack the rigid cell wall that protects bacteria and plants cells from excessive expansion of their bilayer. Instead, a cortical cytoskeleton (CSK) provides a structural framework that allows the animal cell to maintain a stable excess membrane area (i.e., for its volume occupied by a sphere) in the form of membrane folds, ruffles, and microvilli. This excess membrane provides an immediate membrane reserve that may protect the bilayer from sudden changes in bilayer tension. Contractile elements within the CSK may locally slacken or tighten bilayer tension to regulate mechanosensitivity, whereas membrane blebbing and tight seal patch formation, by using up membrane reserves, may increase membrane mechanosensitivity. In specific cases, extracellular and/or CSK proteins (i.e., tethers) may transmit mechanical forces to the process (e.g., hair cell MG channels, MS intracellular Ca(2+) release, and transmitter release) without increasing tension in the lipid bilayer.

In mechanosensitive (MS) channels, gating is initiated by changes in intra-bilayer pressure profiles originating from bilayer deformation. Here we evaluated two physical mechanisms as triggers of MS channel gating: the energetic cost of protein-bilayer hydrophobic mismatches and the geometric consequences of bilayer intrinsic curvature. Structural changes in the Escherichia coli large MS channel (MscL) were studied under nominally zero transbilayer pressures using both patch clamp and EPR spectroscopic approaches. Changes in membrane intrinsic curvature induced by the external addition of lysophosphatidylcholine (LPC) generated massive spectroscopic changes in the narrow constriction that forms the channel 'gate', trapping the channel in the fully open state. Hydrophobic mismatch alone was unable to open the channel, but decreasing bilayer thickness lowered MscL activation energy, stabilizing a structurally distinct closed channel intermediate. We propose that the mechanism of mechanotransduction in MS channels is defined by both local and global asymmetries in the transbilayer pressure profile at the lipid-protein interface.

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherchia coli and have discovered pressure-activated ion channels. The channels have the following properties: (t) activation by slight positive or negative pressure; (it) voltage dependence; (ifi) large conductance; (iv) selectivity for anions over cations; (v) dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.Ion channels are gated protein pores found in biological membranes; these channels regulate many cellular interactions with the environment, including responses to hormonal, neuronal, and sensory stimuli (1). Ion channels have been studied in animals, plants, and microorganisms (2-4). In bacteria, in vivo channel activity has not been demonstrated, although the activity of isolated channel proteins has been measured in artificial membranes (5, 6).The patch-clamp technique allows recording of current through individual ion channels in the native membrane by sucking the membrane onto a recording pipette to form a tight (gigaohm) electrical seal (7). This method has been used to study single channels in vivo in many eukaryotic cells, and it has demonstrated that the large currents measured across the membranes of a whole cell are really composed ofmany small currents passing through individual channels.The lower limit to the diameter ofthe patch-pipette opening is about 1 ,um (1); this precludes measurement ofion channels in bacteria directly. Cells of Escherichia coli, however, can become giant spheroplasts when grown in the presence of chemicals such as mecillinam to prevent cell wall (peptidoglycan) synthesis, and membrane potential has been measured in such spheroplasts by conventional electrophysiology (8). Giant spheroplasts can also be formed by growth of cells in the presence of cephalexin to prevent cell division and form filamentous "snakes"; these snakes can then be treated with lysozyme and EDTA to dissolve the cell wall (the spheroplasts can revert to normal form when returned to growth medium in the absence of these chemicals) (9). We used this latter method to make spheroplasts with a diameter of -6 ,.m. We demonstrate here the application of in vivo patch-clamp recording to such giant spheroplasts. This method should be generally applicable to any bacterial species.We discovered that a low positive or negative pressure (tens of millimeters of mercury; 1 mm Hg = 133 Pa) applied to the spheroplast membrane activates ion channels. This pressure could be caused by an osmotic difference of as little as a few milliosmolar across the membrane. We believe that these channels may allow E. coli to detect and to respond to small osmotic changes in the surrounding medium. The preliminary work has been reported in abstract form (10). MATERIALS AND METHODSMaterials. Organic components of the growth medium were purchased from Difco. Tris was purchased from Boehringer Mannheim; other salts and chemicals for preparation of...

All cellular organisms respond to vibration, touch, gravity or changes in osmolarity, although the molecules on which such mechanosensations depend are unknown. Candidates include certain channels that gate in response to membrane stretch. Patch-clamp experiments with Escherichia coli envelope have revealed a mechanosensitive channel with very large conductance (MscL) and one with a smaller conductance (MscS) which may be important in osmoregulation. Here we have solubilized and fractionated the envelope, reconstituted the MscL activity in vitro, and traced it to a small protein, whose gene, mscL, we then cloned. Insertional disruption of mscL removes the channel activity, whereas re-expression of mscL borne on an expression plasmid restores it. MscL-channel activities were observed in material from a cell-free expression system with mscL as the only template. The mscL nucleotide sequence predicts a unique protein of only 136 amino acids, with a highly hydrophobic core and very different from porins or other known proteins.

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 mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na(+), K(+), Ca(2+) and Mg(2+)) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (>or=10(7)MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (M(r) 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

The conversion of mechanical force to chemical signals is critical for many biological processes, including the sense of touch, pain, and hearing. Mechanosensitive ion channels play a key role in sensing the mechanical stimuli experienced by various cell types, and are present in bacteria to mammals. Bacterial mechanosensitive channels are characterized thoroughly, but less is known about their counterparts in vertebrates. Piezos have been recently established as ion channels required for mechanotransduction in disparate cell types in vitro and in vivo. Overexpression of Piezos in heterologous cells gives rise to large mechanically activated currents; however, it is unclear whether Piezos are inherently mechanosensitive or rely on alternate cellular components to sense mechanical stimuli. Here we show that mechanical perturbations of the lipid bilayer alone are sufficient to activate Piezo channels, illustrating their innate ability as molecular force transducers.