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...

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