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...

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35–55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

A 21 year old male student presented in 1980 as an Olympic athlete with a 12 year history of jaundice, pallor, and darkened urine induced by the atraumatic exercise of swimming (1). Physical examination at that time was remarkable only for moderate scleral icterus without hepatosplenomegaly. Hematological examination revealed moderate macrocytosis (MCV 102 fL) without anemia (Hct 50%, Hb 17 g/dL, 9% reticulocytes). The peripheral blood smear showed occasional target cells. Red cell osmotic fragility was decreased. Red cell Na content was increased and K content was decreased, with reduced total monovalent ion content. Passive red cell permeability of both Na and K were increased. A supervised 2.5 hr swimming workout increased free plasma Hb from <5 to 45 mg/dL and decreased serum haptoglobin from 25 to 6 mg/dL. The post-exercise urine sediment was remarkable for hemosiderin-laden tubular epithelial cells, without frank hemoglobinuria. The circulating 15 day erythrocyte half-life measured after 6 days without exercise was further shortened to 12 days after resumption of twice-per-day swimming workouts for 1 week. The patient’s red cells were hypersensitive to in vitro shear stress applied by cone-plate viscometer.

The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection.

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy 1 gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COXmediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5 e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E were predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.