Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brainderived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca 2+ , a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells. (5) and that the mechanical properties of the culturing environment before transplantation can influence the outcome of in vivo stem cell transplants (6). Hence, a molecular and mechanistic understanding of how stem cells process mechanical cues and how this processing results in downstream signaling events and ultimately in fate decisions is needed for greater control over the fate of transplanted cells.Studies in mesenchymal and neural stem cells have revealed the involvement of focal adhesion zones and cytoskeletal proteins, such as integrins, nonmuscle myosin II (7), Rho GTPases (8-10), and vinculin (11), that participate in the generation of cellular traction forces. Recent work also has identified the nucleoskeletal protein lamin-A (12) and the transcriptional coactivators Yap (Yesassociated protein) and Taz (transcriptional coactivator with PDZbinding motif) (13) in mechanotransduction in mesenchymal stem cells. However, the mechanisms by which mechanical cues detected by cellular traction forces are transduced to downstream intracellular pathways of differentiation remain unclear.Ion channels are involved, directly or indirectly, in the transduction of all forms of physical stimuli-including sound, light, temperature, mechanical force, and even gravity-into intracellular signaling pathways. Hence, we wondered whether ion channels could be involved in transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification. In particular, we focused here on cationic stretch-activated channels (SACs) because they are known to detect mechanical forces with high sensitivity and broad dynamic range and because they are permeable to Ca 2+ , an important second messenger implicated in cell fate (14,15). We examined the role of SACs in neural stem cells, for which mechanical cues influence specification alo...

In voltage-gated channels, ions flow through a single pore located at the interface between membrane-spanning pore domains from each of four subunits, and the gates of the pore are controlled by four peripheral voltage-sensing domains. In a striking exception, the newly discovered voltage-gated Hv1 proton channels lack a homologous pore domain, leaving the location of the pore unknown. Also unknown are the number of subunits and the mechanism of gating. We find that Hv1 is a dimer and that each subunit contains its own pore and gate, which is controlled by its own voltage sensor. Our experiments show that the cytosolic domain of the channel is necessary and sufficient for dimerization and that the transmembrane part of the channel is functional also when monomerized. The results suggest a mechanism of gating whereby the voltage sensor and gate are one and the same.

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.

Voltage-gated ion channels sense voltage by shuttling arginine residues located in the S4 segment across the membrane electric field. The molecular pathway for this arginine permeation is not understood, nor is the filtering mechanism that permits passage of charged arginines but excludes solution ions. We find that substituting the first S4 arginine with smaller amino acids opens a high-conductance pathway for solution cations in the Shaker K(+) channel at rest. The cationic current does not flow through the central K(+) pore and is influenced by mutation of a conserved residue in S2, suggesting that it flows through a protein pathway within the voltage-sensing domain. The current can be carried by guanidinium ions, suggesting that this is the pathway for transmembrane arginine permeation. We propose that when S4 moves it ratchets between conformations in which one arginine after another occupies and occludes to ions the narrowest part of this pathway.

Neurons transmit information through electrical signals generated by voltage-gated ion channels. These channels consist of a large superfamily of proteins that form channels selective for potassium, sodium, or calcium ions. In this review we focus on the molecular mechanisms by which these channels convert changes in membrane voltage into the opening and closing of "gates" that turn ion conductance on and off. An explosion of new studies in the last year, including the first X-ray crystal structure of a mammalian voltage-gated potassium channel, has led to radically different interpretations of the structure and molecular motion of the voltage sensor. The interpretations are as distinct as the techniques employed for the studies: crystallography, fluorescence, accessibility analysis, and electrophysiology. We discuss the likely causes of the discrepant results in an attempt to identify the missing information that will help resolve the controversy and reveal the mechanism by which a voltage sensor controls the channel's gates.

Piezo channels transduce mechanical stimuli into electrical and chemical signals to powerfully influence development, tissue homeostasis, and regeneration. Studies on Piezo1 have largely focused on transduction of “outside-in” mechanical forces, and its response to internal, cell-generated forces remains poorly understood. Here, using measurements of endogenous Piezo1 activity and traction forces in native cellular conditions, we show that cellular traction forces generate spatially-restricted Piezo1-mediated Ca 2+ flickers in the absence of externally-applied mechanical forces. Although Piezo1 channels diffuse readily in the plasma membrane and are widely distributed across the cell, their flicker activity is enriched near force-producing adhesions. The mechanical force that activates Piezo1 arises from Myosin II phosphorylation by Myosin Light Chain Kinase. We propose that Piezo1 Ca 2+ flickers allow spatial segregation of mechanotransduction events, and that mobility allows Piezo1 channels to explore a large number of mechanical microdomains and thus respond to a greater diversity of mechanical cues.

SUMMARYIn voltage-gated sodium, potassium, and calcium channels the functions of ion conduction and voltage sensing are performed by two distinct structural units: the pore domain and the voltage-sensing domain (VSD). In the Hv1 voltage-gated proton channel, the VSD has the remarkable property of performing both functions. Hv1 was recently found to dimerize and to form channels made of two pores. However, the channels were also found to function when dimerization was prevented, raising a question about the functional role of dimerization. Here we show that the two subunits of the Hv1 dimer influence one another during gating, with positive cooperativity shaping the response to voltage of the two pores. We also find that the two voltage sensors undergo conformational changes that precede pore opening and that these conformational changes are allosterically coupled between the two subunits. Our results point to a major role of dimerization in the modulation of Hv1 activity.

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.