Slower Piezo1 Inactivation in Dehydrated Hereditary Stomatocytosis (Xerocytosis)
2013; Elsevier BV; Volume: 105; Issue: 4 Linguagem: Inglês
10.1016/j.bpj.2013.07.018
ISSN1542-0086
AutoresSophie Demolombe, Fabrice Duprat, Éric Honoré, Amanda Patel,
Tópico(s)Literature, Magical Realism, García Márquez
ResumoThe molecular basis for nonselective stretch-activated cationic channels (SACs) has long remained a mystery and is highly controversial (1Nilius B. Honoré E. Sensing pressure with ion channels.Trends Neurosci. 2012; 35: 477-486Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Recently, in a tour de force, by using a knock-down strategy, the Patapoutian laboratory identified two novel proteins, Piezo1 (2521 amino acids) and Piezo2 (2752 amino acids), which are necessary and sufficient for SAC activity (2Coste B. Mathur J. Patapoutian A. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1437) Google Scholar). These proteins are made of ∼30–40 transmembrane segments with both amino and carboxy termini predicted to be intracellular (Fig. 1). Both isoforms are widely expressed, although with some notable exceptions (as, for instance, dorsal root ganglion neurons in which only Piezo2 is found (2Coste B. Mathur J. Patapoutian A. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1437) Google Scholar)). Antibody staining and mass spectrometry analysis indicated that Piezo1 is found at the plasma membrane of red blood cells (RBCs) (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar). Homotetrameric assembly has been demonstrated for Piezo1 (1.2 MDa). Purified Piezo1 was reconstituted into artificial bilayers, but there was no demonstration of stretch sensitivity (4Coste B. Xiao B. Patapoutian A. et al.Piezo proteins are pore-forming subunits of mechanically activated channels.Nature. 2012; 483: 176-181Crossref PubMed Scopus (622) Google Scholar). These important results indicate that Piezo1 is a pore forming subunit. Whether each monomer can conduct ions, or ion permeation requires a tetramer, is still unknown. Upon expression in transfected cells, Piezo1 yields large mechano-activated currents (2Coste B. Mathur J. Patapoutian A. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1437) Google Scholar, 5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar). SAC activity can be elicited in the whole cell configuration by indentation of the cell with a micromanipulated glass stylus. Similarly, SAC activity is recorded in the cell-attached or excised patch configurations and the kinetics studied with a fast pressure clamp. The most remarkable difference between whole-cell and cell-attached patch recordings was the differential sensitivity to actin disruption by cytochalasin D (5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar). Cell-attached Piezo1 currents were not affected, whereas whole-cell configuration was strongly inhibited by cytochalasin D (5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar). Thus, force transmission to Piezo1 induced by whole-cell indentation may require the actin cytoskeleton. In the cell-attached configuration, both positive and negative pressures activate Piezo1, indicating that tension (and not curvature) is gating the channel (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). Piezo1 activity is inhibited, as previously demonstrated for native SACs, by the spider peptide GsMTx-4, which acts as a gating modifier (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar, 7Suchyna T.M. Tape S.E. Gottlieb P.A. et al.Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers.Nature. 2004; 430: 235-240Crossref PubMed Scopus (245) Google Scholar). Currents recorded in either Piezo1- or Piezo2-transfected cells are characterized by a time-dependent inactivation at constant pressure with a monoexponential time course (2Coste B. Mathur J. Patapoutian A. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1437) Google Scholar, 5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar). Using a protocol of dual pressure pulses, it was demonstrated that Piezo1 channels inactivate rather than adapt (5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar). The channel kinetics are well fit with a linear three-state model with one open, one closed, and one inactivated state (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). Several dominant missense mutations of Piezo1 have been recently shown to be associated with dehydrated hereditary stomatocytosis (DHS; also called Xerocytosis), a condition where red blood cells show increased cation permeability and shrinkage (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar, 8Zarychanski R. Schulz V.P. Gallagher P.G. et al.Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary Xerocytosis.Blood. 2012; 120: 1908-1915Crossref PubMed Scopus (285) Google Scholar, 9Albuisson J. Murthy S.E. Patapoutian A. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (222) Google Scholar). RBCs from DHS patients are characterized by higher Na+ content and decreased K+ content (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar). In line with these clinical observations, cell-attached patch recording of RBCs from DHS patients showed increased constitutive channel activity which was blocked by GsMTx4, suggesting a role for Piezo1 (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar). Interestingly, although Piezo1 is widely expressed in many cell types, mutations only (apparently) affect RBC physiology. Perhaps RBCs are more sensitive to disruption of ionic homeostasis, or, because of high mechanical stress in the blood stream (laminar and turbulent shear stress), RBCs are more strongly affected by those mutations. The amino acids mutated in Xerocytosis are located in two regions: between residues 718 and 1358, and the carboxy-terminal region starting at residue 2000 (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar, 8Zarychanski R. Schulz V.P. Gallagher P.G. et al.Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary Xerocytosis.Blood. 2012; 120: 1908-1915Crossref PubMed Scopus (285) Google Scholar, 9Albuisson J. Murthy S.E. Patapoutian A. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (222) Google Scholar). These genetic findings suggest that both domains may be important for the function of Piezo1. Three groups (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar, 6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar, 9Albuisson J. Murthy S.E. Patapoutian A. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (222) Google Scholar) including Bae et al. (10Bae C. Gottlieb P.A. Sachs F. Human Piezo1: removing inactivation with two mutations that each slow inactivation.Biophys. J. 2013; 105: 880-886Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) have shown, almost at the same time, that these pathogenic mutations result in a gain-of-function in Piezo1. The pathogenic mutation R2456H, predicted to be located on the proximal side of the cytosolic carboxy terminal end (Fig. 1), slows inactivation (by approximately threefold) (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar, 9Albuisson J. Murthy S.E. Patapoutian A. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (222) Google Scholar). In addition to these changes, a leftward shift of the pressure-effect curve (by ∼10 mm Hg) was demonstrated, suggesting that the mutant channel is prestressed (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). The conservative mutation R2456K (not described as pathogenic) gave the same phenotype, indicating that residue charge is not involved. GsMTx4 inhibited the pathogenic mutants and ionic selectivity was not altered, suggesting that the pore of the channel was not modified (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). Another pathogenic mutant M2225R, predicted to be located at the extracellular side of the carboxy terminal end (Fig. 1), produced a similar effect, although with a milder slowing of inactivation (∼1.5-fold) (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar, 9Albuisson J. Murthy S.E. Patapoutian A. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (222) Google Scholar). Interestingly, a C-terminal truncated mutant (G2218 stop) also delayed inactivation (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). Thus, the carboxy terminal domain of Piezo1, which is mutated in DHS, plays a key role in Piezo1 inactivation. The gain-of-function in Piezo1 correlates with dehydration of RBCs (3Andolfo I. Alper S.L. Iolascon A. et al.Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.Blood. 2013; 121 (S1–S12): 3925-3935Crossref PubMed Scopus (199) Google Scholar, 6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar, 9Albuisson J. Murthy S.E. Patapoutian A. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (222) Google Scholar). Increased Piezo1 conductance by increased open time will result in increased Na+ and Ca2+ influx, as well as K+ efflux. Ca2+ inflow may, in turn, stimulate the activity of BK channels (Gardos) amplifying K+ efflux and water loss. In this issue of Biophysical Journal, Bae et al. (10Bae C. Gottlieb P.A. Sachs F. Human Piezo1: removing inactivation with two mutations that each slow inactivation.Biophys. J. 2013; 105: 880-886Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) further elegantly demonstrates that when both mutants (M2225R and R2456K) are introduced at the same time, inactivation of Piezo1 is absent (10Bae C. Gottlieb P.A. Sachs F. Human Piezo1: removing inactivation with two mutations that each slow inactivation.Biophys. J. 2013; 105: 880-886Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Moreover, the midpoint of the Boltzmann gating relationship was shifted toward lower pressures (by ∼30 mm Hg), although the slope sensitivity was unchanged. These findings indicate that prestress favoring the open state is enhanced, but that the energy difference between closed and open states (i.e., the dimensional changes of the channel, estimated to be ∼20 nm2) was not altered. Moreover, it shows that activation is independent of inactivation. Prestress on Piezo1 wild-type (WT) can be achieved with hypoosmotic swelling (5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar), but remarkably, the double mutant is not influenced by swelling. This implies that the channel is already maximally prestressed. Increased prestress in the direction favoring opening makes the channel feel like it is being pulled on. Thus, when the channel is prestressed this way, it takes less external pressure to open it. Conversely, GsMTx4 prestresses the channel in the opposite direction, favoring closure, and therefore it takes more pressure to open it (channel is inhibited at a given pressure) (7Suchyna T.M. Tape S.E. Gottlieb P.A. et al.Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers.Nature. 2004; 430: 235-240Crossref PubMed Scopus (245) Google Scholar). Deactivation of the double mutant was also dramatically slowed. The protein domains involved in the closing of the channel (presumably the C-terminal domain), by inactivation or deactivation, may be related. Finally, latency for opening in response to pressure was increased in the double mutant as compared to Piezo1 WT. Inactivation of WT Piezo1 is labile upon repetitive mechanical stimulation (5Gottlieb P.A. Bae C. Sachs F. Gating the mechanical channel Piezo1: a comparison between whole-cell and patch recording.Channels (Austin). 2012; 6: 282-289Crossref PubMed Scopus (121) Google Scholar). Loss of inactivation was seen for many channels at the same time, suggesting that channels are located in fragile domains. These domains can be ruptured by excessive mechanical stress, although clearly below lytic tension of the bilayer (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). Thus, it is likely that a physical domain such as a lipid raft, caveolus, or protein corral affects Piezo1 inactivation by influencing channel-channel (or monomer-monomer) interactions (6Bae C. Gnanasambandam R. Gottlieb P.A. et al.Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.Proc. Natl. Acad. Sci. USA. 2013; 110: E1162-E1168Crossref PubMed Scopus (207) Google Scholar). When microdomains containing groups of channels are fractured, channels (or monomers) may diffuse away and then inactivate more slowly. Pathogenic mutations in the C-terminal domain could allow easier dissociation between channels, which may result in a slower inactivation. In line with this model, the slow deactivation of the double mutant may represent the slow reaggregation of channels. Interestingly, the cationic current (called pSickle) associated with sickle cell disease, in which RBCs are abnormally shaped due to hemoglobin aggregates, is inhibited by GsMTx-4, suggesting that Piezo1 channels might be activated by pressing of aggregates against the membrane causing an increase in local tension (11Lew V.L. Bookchin R.M. Ion transport pathology in the mechanism of sickle cell dehydration.Physiol. Rev. 2005; 85: 179-200Crossref PubMed Scopus (131) Google Scholar). Altogether, these findings indicate that disrupted mechanotransduction of RBCs is anticipated to greatly contribute to stomatocytosis. The work of Bae et al. (10Bae C. Gottlieb P.A. Sachs F. Human Piezo1: removing inactivation with two mutations that each slow inactivation.Biophys. J. 2013; 105: 880-886Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) bring new (to our knowledge) and important findings to better understand how Piezo1 is gated and modified by pathogenic mutations causing DHS. Human PIEZO1: Removing InactivationBae et al.Biophysical JournalAugust 20, 2013In BriefPIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. Full-Text PDF Open Archive
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