Bimolecular membranes are formed from two lipid monolayers at an air-water interface by the apposition of their hydrocarbon chains when an aperture in a Teflon partition separating two aqueous phases is lowered through the interface. Lipid bilayers, which are thought to be the basic structural element of cell membranes, account for many of their properties. They can be assembled from lipids either as small vesicles (1) or as single planar structures that separate two aqueous phases (2). Both models complement each other, and each has its own advantages and shortcomings. The spherical bilayers allow flux measurements with relative ease, and the absence of hydrocarbon solvent may be a factor aiding the incorporation of membrane proteins for functional reconstitutions (3-5). However, their inner compartment is small and inaccessible to chemical manipulation and electrical measurements. In planar bilayers, both compartments are easily accessible, but their mode of formation and the presence of hydrocarbon solvent may be responsible for reported failures to incorporate large membrane proteins. In addition, their electrical capacity is considerably lower than that of cell membranes, implying a different structure or thickness of the dielectric region.For these reasons the formation of planar bilayers without the aid of a hydrocarbon solvent would be desirable. We report here the formation of planar bilayers separating two aqueous phases, in the absence of hydrocarbon solvent, by the hydrophobic apposition of two lipid monolayers at an airwater interface, by a modification of the method used by Takagi, Azuma, and Kishimoto (6) to form "rhodopsin membranes." It will be shown that the electrical capacity of these bilayers exactly matches that of biological membranes, and that the system allows the formation of asymmetric membranes; eventually, this technique may aid in the incorporation of membrane proteins into the lipid bilayer. MATERIALS AND METHODSThe following chemicals were used: glyceroldioleate ( The membranes were formed initially with a modified version of the apparatus described by Takagi (9) (see Fig. la Fig. la). The septum is sealed with silicone grease to the walls of the trough and insulates the two water compartments electrically. It can be rmoved by a motor at a preset speed downwards, so that the aperture moves from above to below the water surface. The troughs and septum were made from Teflon. The aperture in the thin Teflon film was formed either by an electrically heated platinum wire, which was ground to a sharp point, or by a punch made from a tuberculin-syringe needle by beveling its wall. In a simplified version of this method, the thin Teflon film with the aperture was clamped between two halves of a trough and kept stationary. The membrane was formed by filling the two compartments with water or saline to below the aperture and, after spreading a lipid monolayer on each side, raising first one, then the other water level slowly above the aperture by gravity flow.It is important that th...

Mechanotransduction plays a crucial role in physiology. Biological processes including sensing touch and sound waves require yet unidentified cation channels that detect pressure. Mouse piezo1 (mpiezo1) and mpiezo2 induce mechanically activated cationic currents in cells; however, it is unknown if piezos are pore-forming ion channels or modulate ion channels. We show that Drosophila piezo (dpiezo) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. mpiezo1 assembles as a ~1.2 million-Dalton homo-oligomer, with no evidence of other proteins in this complex. Finally, purified mpiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium red-sensitive ion channels. These data demonstrate that piezos are an evolutionarily conserved ion channel family involved in mechanotransduction.

A lattice model of protein folding is developed to distinguish between amino acid sequences that do and do not fold into unique conformations. Although Monte Carlo simulations provide insights into the long-time processes involved in protein folding, these simulations cannot systematically chart the conformational energy surface that enables folding. By assuming that protein folding occurs after chain collapse, a kinetic map of important pathways on this surface is constructed through the use of an analytical theory of probability flow. Convergent kinetic pathways, or "folding funnels," guide folding to a unique, stable, native conformation. Solution of the probability flow equations is facilitated by limiting treatment to diffusion between geometrically similar collapsed conformers. Similarity is measured in terms of a reconfigurational distance. Two specific amino acid sequences are deemed foldable and nonfoldable because one gives rise to a single, large folding funnel leading to a native conformation and the other has multiple pathways leading to several stable conformers. Monte Carlo simulations demonstrate that folding funnel calculations accurately predict the fact of and the pathways involved in folding-specific sequences. The existence of folding funnels for specific sequences suggests that geometrically related families of stable, collapsed conformers fulfill kinetic and thermodynamic requirements of protein folding.

Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ∼3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function.DOI: http://dx.doi.org/10.7554/eLife.07369.001

Excitotoxic neuronal death, associated with neurodegenerative disorders and hypoxic insults, results from excessive exposure to excitatory neurotransmitters. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from overstimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondria are primary targets in excitotoxicity by confocal imaging of intracellular Ca2+ ([Ca2+]i) and mitochondrial membrane potential (delta psi) on cultured rat hippocampal neurons. Sustained activation of NMDA receptors (20 min) elicits reversible elevation of [Ca2+]i. Longer activation (50 min) renders elevation of [Ca2+]i irreversible (Ca2+ overload). Susceptibility to NMDA-induced Ca2+ overload is increased when the 20 min stimuli are applied to neurons pretreated with electron transport chain inhibitors, thereby implicating mitochondria in [Ca2+]i homeostasis during excitotoxic challenges. Remarkably, delta psi exhibits prominent and persistent depolarization in response to NMDA, which closely parallels the incidence of neuronal death. Blockade of the mitochondrial permeability transition pore by cyclosporin A allows complete recovery of delta psi and prevents cell death. These results suggest that early mitochondrial damage plays a key role in induction of glutamate neurotoxicity.