Hailey-Hailey disease as an orthodisease of PMR1 deficiency in Saccharomyces cerevisiae

The term orthodisease has recently been introduced to define human disorders in which the pathogenic gene has orthologs in model organism genomes. Here, we describe Hailey-Hailey disease (HHD), a blistering skin disorder caused by haploinsufficiency of ATP2C1 as an orthodisease from a Saccharomyces cerevisiae perspective. ATP2C1 encodes the human secretory pathway Ca(2+)/Mn(2+) ATPase hSPCA1 and is orthologous to the PMR1 gene in S. cerevisiae. hSPCA1 fully complements PMR1 deficiency in yeast and pmr1DeltaS. cerevisiae has proved to be a valuable tool to screen ATP2C1 mutations and address potential pathogenic/pharmacologic mechanisms in HHD. Consequently, this human skin disorder is an ideal example of an orthodisease.     

The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions

Accumulation of Ca(2+) into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and by secretory pathway Ca(2+)-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca(2+), demonstrated its Mn(2+)-transporting activity, showed its Ca(2+)- and Mn(2+)-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of (45)Ca(2+) (K(0.5) = 0.27 microm) or (54)Mn(2+) transport into an A23187-releasable compartment.     

Manganese selectivity of pmr1, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6. Residue Asp778 is essential for cation transport

We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca(2+)- and Mn(2+)-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment M6 of Pmr1. In phenotypic screens of Ca(2+) chelator and Mn(2+) toxicity reported separately (Wei, Y., Chen, J., Rosas, G., Tompkins, D.A., Holt, P.A., and Rao, R. (2000) J. Biol. Chem. 275, XXXX-XXXX), D778A was a loss-of-function mutant apparently defective for transport of both Ca(2+) and Mn(2+), whereas mutant Q783A displayed a differential sensitivity consistent with the selective loss of Mn(2+) transport. We show that mutant D778A is devoid of cation-dependent ATP hydrolytic activity and phosphoenzyme formation from ATP. However, reverse phosphorylation from P(i) is preserved but is insensitive to inhibition by Ca(2+) or Mn(2+) ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca(2+) can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 micrometer, essentially identical to that of wild type (0.07 micrometer). Mn(2+) activation of ATP hydrolysis was half-maximal at 0.02 micrometer in wild type, establishing a normal selectivity profile of Mn(2+) > Ca(2+). Strikingly, Mn(2+)-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 micrometer. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln(783) in M6 may form a critical hydrophobic interaction with Val(335) in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.     

A novel isoform of the secretory pathway Ca2+,Mn(2+)-ATPase, hSPCA2, has unusual properties and is expressed in the brain

Unlike lower eukaryotes, mammalian genomes have a second gene, ATP2C2, encoding a putative member of the family of secretory pathway Ca2+,Mn(2+)-ATPases, SPCA2. Human SPCA2 shares 64% amino acid identity with the protein defective in Hailey Hailey disease, hSPCA1. We show that human SPCA2 (hSPCA2) has a more limited tissue distribution than hSPCA1, with prominent protein expression in brain and testis. In primary neuronal cells, endogenous SPCA2 has a highly punctate distribution that overlaps with vesicles derived from the trans-Golgi network and is thus different from the compact perinuclear distribution of hSPCA1 seen in keratinocytes and nonpolarized cells. Heterologous expression in a yeast strain lacking endogenous Ca2+ pumps reveals further functional differences from hSPCA1. Although the Mn(2+)-specific phenotype of hSPCA2 is similar to that of hSPCA1, Ca2+ ions are transported with much poorer affinity, resulting in only weak complementation of Ca(2+)-specific yeast phenotypes. These observations suggest that SPCA2 may have a more specialized role in mammalian cells, possibly in cellular detoxification of Mn2+ ions, similar to that in yeast. We point to the close links between manganese neurotoxicity and Parkinsonism that would predict an important physiological role for SPCA2 in the brain.     

A Family with Atypical Hailey Hailey Disease- Is There More to the Underlying Genetics than ATP2C1?

The autosomal dominant Hailey Hailey disease (HHD) is caused by mutations in the ATP2C1 gene encoding for human secretory pathway Ca2+/Mn2+ ATPase protein (hSPCA1) in the Golgi apparatus. Clinically, HHD presents with erosions and hyperkeratosis predominantly in the intertrigines. Here we report an exome next generation sequencing (NGS) based analysis of ATPase genes in a Greek family with 3 HHD patients presenting with clinically atypical lesions mainly localized on the neck and shoulders. By NGS of one HHD-patient and in silico SNP calling and SNP filtering we identified a SNP in the expected ATP2C1 gene and SNPs in further ATPase genes. Verification in all 3 affected family members revealed a heterozygous frameshift deletion at position 2355_2358 in exon 24 of ATP2C1 in all three patients. 7 additional SNPs in 4 ATPase genes (ATP9B, ATP11A, ATP2B3 and ATP13A5) were identified. The SNPs rs138177421 in the ATP9B gene and rs2280268 in the ATP13A5 gene were detected in all 3 affected, but not in 2 non affected family members. The SNPs in the ATP2B3 and ATP11A gene as well as further SNPs in the ATP13A5 gene could not be confirmed in all affected family members. One may speculate that besides the level of functional hSPCA1 protein, levels of other ATPase proteins may influence expressivity of the disease and might also contribute, as in this case, to atypical presentations.     

A novel missense mutation of the ATP2C1 gene in a Chinese patient with Hailey-Hailey disease

Benign familial chronic pemphigus (Hailey–Hailey disease, HHD; MIM 169600) is a rare autosomal dominant hereditary disorder characterized by pruritic vesicles, painful erosions and scaly erythematous plaques at the sites of friction and flexures. Mutations in ATP2C1, which encoding the human secretory pathway Ca²⁺/Mn²⁺-ATPase protein 1 (hSPCA1), have been identified as the pathogenic gene of HHD. We found a novel, distinct, heterozygous mutation during study of a Chinese patient with HHD. We identified a C→T transition at nucleotide 1235 (p.Thr352IIe), in exon 13 of ATP2C1. This observation would be useful for genetic counseling and prenatal diagnosis for affected families and in expanding the repertoire of ATP2C1 mutations underlying HHD.     

Mutational analysis of the role of Glu309 in the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle.

Site-specific mutagenesis was used to analyse the role of the residue, Glu309, in the function of the Ca(2+)-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 microM Ca2+ was unable to prevent phosphorylation from Pi, consistent with previous observations on the Ca(2+)-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476-478]. At neutral pH, however, micromolar concentrations of Ca2+ were sufficient to inhibit phosphorylation of the Glu309----Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca2+ site was relatively intact in this mutant. The Glu309----Lys mutant was unable to form a phosphoenzyme from ATP at all Ca2+ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu309----Ala mutant occurred at 12.5 mM Ca2+, but not at Ca2+ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E2P intermediate in the Glu309----Lys mutant. A less pronounced stabilization of E2P was observed with the Glu309----Ala mutant, suggesting a possible role of the charge at the position of Glu309 in phosphoenzyme hydrolysis.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

Changes in affinity of Na+- and K+-transport ATPase for divalent cations during its reaction sequence

Previous experiments (Fukushima, Y., and Post, R.L. (1978) J. Biol. Chem. 253, 6853-6862) demonstrated that the Ca x phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase gradually becomes stable after dissociation of Ca2+ in the presence of a chelating agent such as 1,2-cyclohexylenedinitrilo-tetraacetic acid. In the present study, we investigated whether the ADP- and K+-sensitive forms of the Ca x phosphoenzyme show different affinities for divalent cations. Our findings were as follows. (a) As the concentraion of Na+ was increased during phosphorylation of the enzyme with ATP at pH 7.4 and 0 degrees C, both the sensitivity to ADP and the amount of calcium-free phosphoenzyme increased in parallel. (b) For this Na+-dependent change, kidney enzyme required higher concentrations of Na+ than did brain enzyme. (c) In addition, the rate of dissociation of Ca2+ from the ADP-sensitive Ca x phosphoenzyme was faster than that from the K+-sensitive phosphoenzyme. It was thus concluded that Ca2+ binds to the ADP-sensitive phosphoenzyme less tightly than to the K+-sensitive phosphoenzyme.     

Functional expression in yeast of the human secretory pathway Ca(2+), Mn(2+)-ATPase defective in Hailey-Hailey disease.

The discovery and biochemical characterization of the secretory pathway Ca(2+)-ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens. In yeast, PMR1 has been shown to function as a high affinity Ca(2+)/Mn(2+) pump and has been localized to the Golgi compartment where it is important for protein sorting, processing, and glycosylation. However, little is known about PMR1 homologues in higher organisms. Loss of one functional allele of the human gene, hSPCA1, has been linked to Hailey-Hailey disease, characterized by skin ulceration and improper keratinocyte adhesion. We demonstrate that expression of hSPCA1 in yeast fully complements pmr1 phenotypes of hypersensitivity to Ca(2+) chelators and Mn(2+) toxicity. Similar to PMR1, epitope-tagged hSPCA1 also resides in the Golgi when expressed in yeast or in chinese hamster ovary cells. (45)Ca(2+) transport by hSPCA1 into isolated yeast Golgi vesicles shows an apparent Ca(2+) affinity of 0.26 microm, is inhibitable by Mn(2+), but is thapsigargin-insensitive. In contrast, heterologous expression of vertebrate sarcoplasmic reticulum and plasma membrane Ca(2+)-ATPases in yeast complement the Ca(2+)- but not Mn(2+)-related phenotypes of the pmr1-null strain, suggesting that high affinity Mn(2+) transport is a unique feature of the secretory pathway Ca(2+)-ATPases.     

Functional consequences of alterations to Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum with their corresponding amides. These residues are predicted to lie in the transmembrane domain and have been suggested as oxygen ligands for Ca2+ binding at high affinity sites (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). The Glu309----Gln and Asp800----Asn mutants were unable to form a phosphoenzyme from ATP at the Ca2+ concentrations examined (up to 12.5 mM), whereas the Glu771----Gln mutant phosphorylated from ATP at 2.5 mM Ca2+. In all three mutants, Ca2+ at concentrations well below 12.5 mM prevented or inhibited phosphorylation with Pi, suggesting that at least one calcium-binding site was functioning in each mutant. In the mutants Glu309----Gln and Glu771----Gln, the ADP-insensitive phosphoenzyme intermediate was unusually stable, as indicated by a very low rate of dephosphorylation observed in kinetic experiments and by an increased apparent affinity for Pi determined in equilibrium phosphorylation experiments. These data indicate a central role of Glu309 and Glu771 in the energy-transducing conformational changes and/or in the activation of phosphoenzyme hydrolysis.     

Desensitization to Mg2+ of the phosphoenzyme in sarcoplasmic reticulum Ca2+-ATPase by the addition of a nonionic detergent and the restoration of sensitivity after its removal

Sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle were solubilized with a high concentration of dodecyl octaethyleneglycol monoether (C12E8) and the kinetic properties of the Ca2+,Mg2+-dependent ATPase [EC 3.6.1.3] were studied. The following results were obtained: 1. SR ATPase solubilized in C12E8 retains high ability to form phosphoenzyme ([EP] = 4--5 mol/10(6) g protein) for at least two days in the presence of 5 mM Ca2+, 0.5 M KCl, and 20% glycerol at pH 7.55. 2. The ATPase activity was dependent on both Mg2+ and Ca2+. However, the rate of E32P decay after the addition of unlabeled ATP was independent of Mg2+. 3. Most of the EP formed in the absence of Mg2+ was capable of reacting with ADP to form ATP in the backward reaction. However, in the presence of 5 mM Mg2+, the amount of ATP formed was markedly reduced without loss of the reactivity of the EP with ADP. 4. The removal of C12E8 from the ATPase by the use of Bio-Beads resulted in the full restoration of the Mg2+ dependency of the EP decomposition. 5. These results strongly suggest that in the case of SR solubilized with a high concentration of C12E8 the decomposition of phosphoenzyme is Mg2+ independent and ATP is mainly hydrolyzed through Mg2+-dependent decomposition of an enzyme-ATP complex, which is in equilibrium with phosphoenzyme and ADP.     

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of calcium

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of Ca2+ was studied. At pH 6.0, 10 degrees C and in the absence of K+, the enzyme displays a very low velocity of ATP hydrolysis. Addition of up to 15% dimethyl sulfoxide increased this velocity severalfold (from 5-18 nmol of Pi X mg of protein-1 X h-1) and then decreased at higher solvent concentrations. Dimethyl sulfoxide increased both enzyme phosphorylation from ATP and the affinity for this substrate. Maximal levels of 1.0-1.2 nmol of EP X mg of protein-1 and apparent KM for ATP of 5 X 10(-6) M were obtained at a concentration of 30% dimethyl sulfoxide. The same preparation under optimal conditions (pH 7.5, 10 microM CaCl2, 100 mM KCl and no dimethyl sulfoxide at 37 degrees C) displays a velocity of ATP hydrolysis between 8 and 12 X 10(5) nmol of Pi X mg of protein-1 X h-1 while the phosphoenzyme levels varied between 3.5 and 4.0 nmol of EP X mg of protein-1. Enzyme phosphorylation from ATP in the absence of Ca2+ always preceded Pi liberation into the assay media. Two different phosphoenzyme species were formed which were kinetically distinguished by their decomposition rates. The observed steady-state velocity of ATP hydrolysis could be accounted for either by the decay of the fast component or by the simultaneous decomposition of both phosphoenzyme species. The hydrolysis of the phosphoenzyme formed in the absence of Ca2+ was KCl-stimulated and ADP-independent. The rate constant of breakdown was equal to that observed for the phosphoenzyme formed in the presence of Ca2+. It is suggested that the rapidly decaying phosphoenzyme (and possibly both rapidly and slowly decaying species) are intermediates in the reaction cycle of Mg2+-dependent ATP hydrolysis of sarcoplasmic reticulum Ca2+-ATPase and may represent a bypass of Ca2+ activation by dimethyl sulfoxide.     

Activation of purinergic P2X receptors inhibits P2Y-mediated Ca2+ influx in human microglia

Purinoceptor (P2X and P2Y) mediated Ca2+ signaling in cultured human microglia was studied using Ca2+ sensitive fluorescence microscopy. ATP (at 100 microM) induced a transient increase in [Ca2+]i in both normal and Ca(2+)-free solution suggesting a primary contribution by release from intracellular stores. This conclusion was further supported by the failure of ATP to cause a divalent cationic influx in Mn2+ quenching experiments. However, when fluorescence quenching was repeated after removal of extracellular Na+, ATP induced a large influx of Mn2+, indicating that inward Na+ current through a non-selective P2X-coupled channel may normally suppress divalent cation influx. Inhibition of Mn2+ entry was also found when microglia were depolarized using elevated external K+ in Na(+)-free solutions. The possibility of P2X inhibition of Ca2+ influx was then investigated by minimizing P2X contributions of purinergic responses using either the specific P2Y agonist, ADP-beta-S in the absence of ATP or using ATP combined with PPADS, a specific inhibitor of P2X receptors. In quenching studies both procedures resulted in large increases in Mn2+ influx in contrast to the lack of effect observed with ATP. In addition, perfusion of either ATP plus PPADS or ADP-beta-S alone caused a significantly enhanced duration (about 200%) of the [Ca2+]i response relative to that induced by ATP. These results show that depolarization induced by P2X-mediated Na+ influx inhibits store-operated Ca2+ entry resulting from P2Y activation, thereby modulating purinergic signaling in human microglia.     

Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strain

PMR1, the Ca2+/Mn2+ ATPase of the secretory pathway in Saccharomyces cerevisiae was the first member of the secretory pathway Ca2+ ATPases (SPCA) to be characterized. In the past few years, pmr1Delta yeast have received more attention due to the recognition that the human homologue of this protein, hSPCA1 is defective in chronic benign pemphigus or Hailey-Hailey disease (HHD). Recent publications have described pmr1Delta S. cerevisiae as a useful model organism for studying the molecular pathology of HHD. Some observations indicated that the high Ca2+ sensitive phenotype of PMR1 defective yeast strains may be the most relevant in this respect. Here we show that the total cellular calcium response of a pmr1Delta S. cerevisiae upon extracellular Ca2+ challenge is decreased compared to the wild type strain similarly as observed in keratinocytes. Additionally, the novel magnesium sensitivity of PMR1 defective yeast is revealed, which appears to be a result of competition for uptake between Ca2+ and Mg2+ at the plasma membrane level. Our findings indicate that extracellular Ca2+ and Mg2+ competitively influence the intracellular Ca2+ homeostasis of S. cerevisiae. These observations may further our understanding of HHD.     

Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle

We relate solution behavior to the crystal structure of the Ca2+ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the gamma-phosphate to reach Asp351 to form a complex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.     

Functional consequences of mutations in the beta-strand sector of the Ca2(+)-ATPase of sarcoplasmic reticulum

Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233 in the reaction mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+ was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+ and a lower cooperativity in Ca2+ binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1 to E2 and E1P to E2P conformational equilibria toward the E1 and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233 were also examined by mutation. Although the side chains of these residues are potential Ca2+ ligands, their replacement did not affect the Ca2+ affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2(+)-binding sites.     

The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].     

P2X and P2Y purinoreceptors mediate ATP-evoked calcium signalling in optic nerve glia in situ

It is known that ATP acts as an extracellular messenger mediating Ca2+ signalling in glial cells. Here, the mechanisms involved in the ATP-evoked increase in glial [Ca2+]i were studied in situ, in the acutely isolated rat optic nerve. ATP and agonists for P2X (a,b-metATP) and P2Y (2MeSATP) purinoreceptors triggered raised glial [Ca2+]i, and there was no significant difference between cells identified morphologically as astrocytes and oligodendrocytes. Dose-response curves indicated that P2Y receptors were activated at nanomolar concentrations, whereas P2X purinoreceptors were only activated above 10 microM. The rank order of potency for several agonists indicated optic nerve glia expressed heterogeneous purinoreceptors, with P2Y1< or = P2Y2/4< or = P2X. The ATP evoked increase in [Ca2+]i was reversibly blocked by the P2X/Y purinoreceptor antagonist suramin (100 microM) and markedly reduced by thapsigargin (10 microM), which blocks IP3-dependent release of Ca2+ from intracellular stores. Removal of extracellular Ca2+ reduced the ATP evoked increase in [Ca2+]i and completely blocked its recovery, indicating that refilling of intracellular stores was ultimately dependent on Ca2+ influx from the extracellular milieu. The results implicate ATP as an important signal in CNS white matter astrocytes and oligodendrocytes in situ, and indicate that metabotropic P2Y purinoreceptors mobilize intracellular Ca2+ at physiological concentrations of ATP, whereas ionotropic P2X purinoreceptors induce Ca2+ influx across the plasmalemma only at high concentrations of ATP, such as occur following CNS injury.