MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (P o) has a steep sigmoidal dependence on T, with a midpoint (T 1/2) of 11.8 dyn/cm. The maximal slope sensitivity of P o/P c was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was ΔE = 18.6 k B T. If the mechanosensitivity arises from tension acting on a change of in-plane area (ΔA), the free energy, TΔA, would correspond to ΔA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest ΔA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to ΔA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Gadolinium ions produce three distinct kinds of block of the stretch-activated (SA) ion channels in Xenopus oocytes: a concentration-dependent reduction in channel open time, a concentration-dependent reduction in open channel current, and a unique, steeply concentration-dependent, reversible inhibition of channel opening. This last effect reduces the probability of a channel being open from about 10(-1) at 5 microM to less than 10(-5) at 10 microM gadolinium. Calcium has effects on open time and current similar to that of gadolinium, but this channel is permeable to calcium and calcium does not completely inhibit channel activity. The availability of a blocker for SA ion channels may help to define their physiological function, and will simplify the use of oocytes as an expression system for ion channels.

We present here a maximal likelihood algorithm for estimating single-channel kinetic parameters from idealized patch-clamp data. The algorithm takes into account missed events caused by limited time resolution of the recording system. Assuming a fixed dead time, we derive an explicit expression for the corrected transition rate matrix by generalizing the theory of Roux and Sauve (1985, Biophys. J. 48:149-158) to the case of multiple conductance levels. We use a variable metric optimizer with analytical derivatives for rapidly maximizing the likelihood. The algorithm is applicable to data containing substates and multiple identical or nonidentical channels. It allows multiple data sets obtained under different experimental conditions, e.g., concentration, voltage, and force, to be fit simultaneously. It also permits a variety of constraints on rate constants and provides standard errors for all estimates of model parameters. The algorithm has been tested extensively on a variety of kinetic models with both simulated and experimental data. It is very efficient and robust; rate constants for a multistate model can often be extracted in a processing time of approximately 1 min, largely independent of the starting values.

SUMMARY1. The membrane of tissue-cultured chick pectoral muscle contains an ionic channel which is activated by membrane stretch. Nicotinic channels and Ca2+_ activated K+ channels are not affected by stretch.2. In 150 mM-external K+ and 150 mM-internal Na+ the channel has a conductance of 70 pS, linear current-voltage relationship between -50 and -140 mV and a reversal potential of + 30 mV. (0) and three closed (C) states. The data can be fitted by the reaction scheme: C1-C2-C33-°O Only the rate constant that governs the C1-C2 transition (k1 2) is stretch-sensitive. None of the rates are voltage-sensitive.4. The rate constant k1,2 varies with the square of the tension as k1,2 = ko . eaT2, where a is a constant describing the sensitivity to stretch and T is the tension. A typical value of a is 0-08 (dyn cm-1)-2.5. Following exposure to cytochalasin B the channel becomes more sensitive to stretch. The stretch-sensitivity constant, a, increases from 0-08 to 2-4 (dyn cm-1)-2.6. The probability of the channel being open is strongly dependent upon the extracellular K+ concentration. With a suction of 2 cmHg the probability increases from 0 004 in normal saline (5 mM-K+) to 0-26 in 150 mM-K+.7. The channel appears to gather force from a large area of membrane (> 3 x 105 A2), probably by a cytochalasin-resistant cytoskeletal network.

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.

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation-selective MSC, defines a new system to study mechanical transduction at the cellular level. Since Piezo1 has electrophysiological properties similar to endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested and found that the peptide targets Piezo1 activity. Extracellular GsMTx4 at μM concentrations reversibly inhibited ~80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive and, as seen with endogenous MSCs, the mirror image D enantiomer inhibited similarly to the L. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 105 M-1s-1 and 0.11s-1 respectively and a KD of ~ 155nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced a ~30mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside out patches. Whole cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off-rate was nearly identical to outside out patches, differences were observed for the on-rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in a high throughput screens for pharmacological agents and diagnostic assays.

Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca 2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca 2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1-or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.A rticular cartilage is a hydrated connective tissue that supports loads and minimizes friction in the diarthrodial joints. It has a highly differentiated extracellular matrix (ECM) composed primarily of type II collagen, the large aggregating proteoglycan, aggrecan, and water. Chondrocytes are the only cells in cartilage and are responsible for maintaining and remodeling cartilage through a homeostatic balance of anabolic and catabolic activities. Under normal physiologic conditions, chondrocytes are exposed to millions of cycles of mechanical loading per year (1). These mechanical signals play an important role in regulating chondrocyte anabolic and biosynthetic activity, as evidenced by cartilage atrophy following periods of disuse or immobilization (2-7). However, under abnormal loading conditions (e.g., due to obesity, trauma, or joint instability), mechanical factors play a critical role in the onset and progression of osteoarthritis (1). Such "injurious" loading has been modeled in vitro using explant culture systems that replicate many of the early cellular and molecular events characteristic of osteoarthritis (8). Osteoarthritis is a painful and debilitating disease of weight-bearing joints that affects over 26 million people in the United States (9) with posttraumatic arthritis being responsible for ∼12% of the incidence of osteoarthritis (10).Despite the critical importance of mechanical loading in health and disease of synovial joints, the mechanisms of mechanotransduction of chondrocytes are not fully understood and are likely to differ under physiologic and pathologic conditions (11)(12)(13)(14). Although many different mechanisms have been shown to be involved in chondrocyte mechanotransduction (13,(15)(16)(17), recent st...

Familial xerocytosis (HX) in humans is an autosomal disease that causes dehydration of red blood cells resulting in hemolytic anemia which has been traced to two individual mutations in the mechanosensitive ion channel, PIEZO1. Each mutation alters channel kinetics in ways that can explain the clinical presentation. Both mutations slowed inactivation and introduced a pronounced latency for activation. A conservative substitution of lysine for arginine (R2456K) eliminated inactivation and also slowed deactivation, indicating that this mutant's loss of charge is not responsible for HX. Fitting the current vs. pressure data to Boltzmann distributions showed that the half-activation pressure, P 1/2 , for M2225R was similar to that of WT, whereas mutations at position 2456 were left shifted. The absolute stress sensitivity was calibrated by cotransfection and comparison with MscL, a well-characterized mechanosensitive channel from bacteria that is driven by bilayer tension. The slope sensitivity of WT and mutant human PIEZO1 (hPIEZO1) was similar to that of MscL implying that the in-plane area increased markedly, by ∼6-20 nm 2 during opening. In addition to the behavior of individual channels, groups of hPIEZO1 channels could undergo simultaneous changes in kinetics including a loss of inactivation and a long (∼200 ms), silent latency for activation. These observations suggest that hPIEZO1 exists in spatial domains whose global properties can modify channel gating. The mutations that create HX affect cation fluxes in two ways: slow inactivation increases the cation flux, and the latency decreases it. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells.mechanical channels | PIEZO1 mutations | channel domains H ereditary xerocytosis (HX) is an autosomal dominant disease characterized by dehydrated red blood cells (RBCs) and mild-to-moderate hemolytic anemia. Two familial HX mutations were identified recently in the gene encoding hPIEZO1, a mechanosensitive ion channel (MSC) (1).Mouse PIEZO1 (mPIEZO1) cloned from Neuro2A cells contains ∼2,500 amino acids predicted to have 24-36 transmembrane domains. Using crosslinking and photobleaching techniques, PIEZO1 was shown to assemble as a homotetramer (2, 3) with no other cofactors. Currently it is not known whether the pore is central to the tetramer (intermolecular) or whether each subunit conducts (intramolecular). mPIEZO1 is a cation-selective channel with a reversal potential near 0 mV. The conductance is ∼70 pS and is reduced to 35 pS by increasing extracellular Mg +2 (3, 4). mPIEZO1, like other cationic MSCs, is inhibited by the peptide GsMTx4 (5) and nonspecifically by ruthenium red (2). Heterologous expression in HEK293 cells is efficient, and mechanical currents can be evoked in whole-cell mode or patches. In cell-attached patches at hyperpolarized potentials, mPIEZO1 activates with ∼30 mmHg of pipette suction and inactivates within ∼30 ms, a rate that slows with depolarization (2-4).To explore the biophysi...