Packing Interactions between Transmembrane Helices Alter Ion Selectivity of the Yeast Golgi Ca2+/Mn2+-ATPase PMR1

Abstract

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions. PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions. The Secretory Pathway Ca2+-ATPases (SPCAs) 1The abbreviations used are: SPCA, secretory pathway Ca2+-ATPase; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; PMCA, plasma membrane Ca2+-ATPase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid. are an emerging family of Mn2+- and Ca2+-transporting P-type ATPases localized to the Golgi apparatus (1Wuytack F. Raeymaekers L. Missiaen L. Eur. J. Physiol. 2003; 446: 148-153Crossref PubMed Scopus (95) Google Scholar, 2Ton V.-K. Mandal D. Vahadji C. Rao R. J. Biol. Chem. 2002; 277: 6422-6427Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Members of this family have been identified in the genomes of diverse organisms, including Saccharomyces cerevisiae and other yeasts (PMR1), Caenorhabditis elegans (PMR1), Drosophila melanogaster (Q9VNR2), and Homo sapiens (ATP2C1). S. cerevisiae PMR1 has distinct inhibitor and transport characteristics, which clearly separate it from the well-studied sarcoendoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+-ATPases (3Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 4Wei Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). Perhaps the most interesting transport characteristic of SPCA pumps is their unique ability to transport Mn2+ ions with high affinity (1Wuytack F. Raeymaekers L. Missiaen L. Eur. J. Physiol. 2003; 446: 148-153Crossref PubMed Scopus (95) Google Scholar, 2Ton V.-K. Mandal D. Vahadji C. Rao R. J. Biol. Chem. 2002; 277: 6422-6427Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Haploinsufficiency of the human ATP2C1 gene for SPCA1 results in Hailey Hailey disease, characterized by acantholysis of the epidermis (5Hu Z. Bonifas J.M. Beech J. Bench G. Shigihara T. Ogawa H. Ikeda S. Mauro T. Epstein E.H. Nat. Genet. 2000; 24: 61-65Crossref PubMed Scopus (446) Google Scholar, 6Sudbrak R. Brown J. Dobson-Stone C. Carter S. Ramser J. White J. Healy E. Dissanayake M. Larregue M. Perrussel M. Lehrach H. Munro C.S. Strachan T. Burge S. Hovnanian A. Monaco A.P. Hum. Mol. Genet. 2000; 9: 1131-1140Crossref PubMed Scopus (262) Google Scholar). Members of the P-ATPase superfamily share common structural and mechanistic properties yet have strikingly different ion specificities that underlie their individual physiological roles. In the reaction cycle of the P-ATPases, the E1 conformation of the pump binds ATP and the cation(s) to be transported with high affinity, followed by transfer of the γ-phosphoryl group of ATP to an invariant aspartate to form an aspartyl-phosphate reaction intermediate. A major change in enzyme conformation to the E2 form ensues, with a large reduction in ion binding affinity and reorientation of the binding pocket resulting in vectorial ion transport (7East J.M. Mol. Membr. Biol. 2000; 17: 189-200Crossref PubMed Scopus (85) Google Scholar). The x-ray crystal structure of SERCA in the E1 conformation, at 2.6-Å resolution, provides a structural definition for two high affinity Ca2+ binding sites enclosed by the transmembrane domains M4, M5, M6, and M8 (8). More recently, a second structure of the enzyme locked in the E2 conformation, was resolved by x-ray crystallography to 3.1 Å (9Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). These structures revealed large domain movements within the cytoplasmic regions as well as twisting and elongation of transmembrane helices involved in cation interactions presumably during transition from the E1 to E2 conformations. The repertoire of cations transported by the P-ATPases includes, but is not limited to, H+, K+, Na+, Ca2+, Cu2+, Zn2+, and Mg2+. To understand the molecular basis for ion specificity, we have used phenotypic screening in yeast to identify mutations that alter selectivity for Ca2+ and Mn2+ in PMR1. Growth of the Δpmr1 null strain is hypersensitive to divalent cation-chelating agents such as BAPTA and EGTA, due to depletion of Ca2+ and Mn2+ from the secretory pathway where they serve essential functions in protein processing, sorting, and glycosylation (10Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J. Davidow L.S. Mao J.M. Moir D.T. Cell. 1989; 58: 133-145Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 11Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar, 12Dürr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (349) Google Scholar). BAPTA toxicity can be overcome by the addition of either Ca2+ or Mn2+ to the medium, indicating that these two ions play largely surrogate roles in supporting growth (13Loukin S. Kung C. J. Cell Biol. 1995; 131: 1025-1037Crossref PubMed Scopus (54) Google Scholar). However, because Ca2+ is present in ∼100-fold excess over Mn2+ in standard yeast media and because Mn2+ is efficiently removed at low chelator concentrations, the observed growth inhibition of pmr1 mutants by BAPTA represents a titration of available Ca2+. Thus, we have previously demonstrated an excellent correlation between loss of Ca2+ transport activity in PMR1 mutants and growth sensitivity to BAPTA (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Loss of Mn2+ transport by PMR1 mutants is known to confer hypersensitivity to Mn2+ toxicity (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Lapinskas P.J. Cunningham K.W. Liu X. Fink G.R. Culotta V. Mol. Cell Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). Although Mn2+ is an essential trace element, excess Mn2+ is toxic and must be sequestered and removed via the secretory pathway by PMR1 (15Lapinskas P.J. Cunningham K.W. Liu X. Fink G.R. Culotta V. Mol. Cell Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). Taken together, growth sensitivities to BAPTA and Mn2+ are rapid and effective means to screen pmr1 mutants for loss of ion transport. sensitivity to BAPTA and Mn2+ is of a change in ion selectivity in the which can be by In a we have the of mutations at at the cytoplasmic interface of transmembrane M6 D. T. Rao R. J. Biol. Cell. 2000; Scholar). residues and could effectively for in the transport of Ca2+ and Mn2+ ions. of side chains and at this to loss of an was unique in a and loss of Mn2+ transport. for a loss of Mn2+ selectivity in the Q783A mutant from the observed loss of Mn2+ inhibition of ATP hydrolysis and and Mn2+ inhibition of from In BAPTA and ATP hydrolysis and were in this mutant (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, D. T. Rao R. J. Biol. Cell. 2000; Scholar). of transmembrane helices M4 and M6 of PMR1 Gln-783 (M6) in to Val-335 and to that mutations at 783 alter ion selectivity by with In this we additional residues in the of Gln-783 and the that the interaction between Gln-783 and Val-335 is critical for Mn2+ transport. and were in yeast and as K.W. Fink G.R. J. Cell Biol. PubMed Scopus Google was used as this strain has activity (3Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). were in the low and for changes in phenotype (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). the PMR1 gene a of the yeast and the of low of PMR1 in at the mutations were into the from which PMR1 was at from the Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). was by a unique to of the PMR1 gene the from into the sites on of PMR1 was by of with and and into which is a of the were by the of mutant PMR1 from the using a that an and a that a The was purified form was with and and into and was to the of the in the most mutations in M2, M4, M5, and M6 were in either a of PMR1 or or in a or into to the and to as previously In the mutagenesis was used to mutations at at 335 was used in with a to mutations at this using the reaction of and L. 1999; Scopus Google Scholar). The was to identify the or substitutions in 335 and to that may have been during in The was used to the in mutations at 335 were with mutations at residue 783 by the into the The of mutations in the was confirmed to the of or assays were in of of was at or for or BAPTA was to the growth at the was to with Growth was by at on a were to in which or BAPTA were and and of or mutant using has been in D. T. Rao R. J. Biol. Cell. 2000; Scholar). of by purified was at in and or was and with to cation that were using the was used to separate from as D. T. Rao R. J. Biol. Cell. 2000; Scholar). from were into of media with to an A of for in a was and yeast were at and for by using were with of in of and in of growth was by at on Homology of of S. cerevisiae PMR1 in the E1 conformation was using by the of PMR1 to which is the 2.6-Å crystal structure of in the E1 C. M. Nomura H. Ogawa H. Nature. 2000; PubMed Scopus Google Scholar). a of PMR1 in the E2 conformation was using from for the SERCA structure (9Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). The in were used for the followed by of the to between M4 and as in The resulting of PMR1 is based on the of and The Ca2+ ion in the was by the Ca2+ ion from of the into the regions of PMR1. The in were by using the and E2 of PMR1 transmembrane helices M4, M5, and Homology models of PMR1 were based on the crystal structures of the E1 and E2 of and the in are to the of the membrane and or in from the cytoplasmic side of the membrane and Gln-783 and Val-335 are in A ion in the E1 conformation has been acid side chains are and based on the of substitutions in this and and Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). to the binding in the E1 structure are in the movements in the cytoplasmic of M4 and M6 that Val-335 and Gln-783 in in the E2 Scanning of to The unique ion selectivity defect of mutant Q783A a mutagenesis of within a based on the E1 crystal structure of the we residues and of M2, and Phe-738 in that could with the side at of these residues was with and Gln-783 near the cytoplasmic interface of M5, we also mutagenesis of residues in the cytoplasmic of transmembrane M4, M5, and were from a low in a yeast strain Ca2+ pumps (3Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google and for of BAPTA and we have ion transport in purified Golgi and ATP hydrolysis in purified enzyme and demonstrated that these properties with growth sensitivity of mutant to BAPTA and Mn2+ toxicity (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, D. T. Rao R. J. Biol. Cell. 2000; Scholar). for BAPTA and Mn2+ for as of at and in transmembrane on PMR1 as by their or to be sensitive to and the was the in M4, loss of in low but was to from a is to have reduction in that can be by of the Phe-738 at the cytoplasmic interface of by either or to a loss in ability to the pmr1 null from or indicating a critical for this We that of residue in M5, with to a loss of (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To the of this residue we mutagenesis at and the resulting substitutions for BAPTA and Mn2+ sensitivity A and different substitutions and the could at this other substitutions to loss of of phenotype on PMR1 of substitutions at on BAPTA and Mn2+ The strain null of Ca2+ pumps was with low or mutant PMR1 the acid substitutions at has a at were in media with BAPTA or Mn2+ Growth is as a of BAPTA or Mn2+ In M6, mutant also near the cytoplasmic interface, a loss of as by of BAPTA and Mn2+ sensitivities in low and high mutant in a to the residue was also mutant was sensitive to Mn2+ to BAPTA of a change in the affinity for Ca2+ and Mn2+ ions in this of in the human SPCA1 has revealed that the Hailey Hailey mutant in M4 to Mn2+ Ca2+ binding affinity L. J. Missiaen L. Raeymaekers L. F. Hovnanian A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is proximal to the in the ion binding be that most substitutions in largely changes in BAPTA and Mn2+ sensitivities in revealed ion selectivity including in M4, and in M6, that are of a of Val-335 is in this In the mutagenesis identified residues near the cytoplasmic interface of M4, M5, and M6 that to be critical for ion transport. In the for a or that residue may be in ion In an we identified or residues in the of transmembrane helices that in ion The results from this and mutagenesis an of the of individual residues the transport of PMR1 have previously demonstrated that PMR1 mutants are in the where they hypersensitive to limited by of structural or (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). was important to mutations in this and to the mutants or loss of were with GFP, and by The mutants a of yeast Golgi and were to Thus, loss of to be to a a at Val-335 or the of on of M4 and M6 we that Gln-783 in M6 a with Val-335 in M4 D. T. Rao R. J. Biol. Cell. 2000; Scholar). To this we a of substitutions at 335 of PMR1 and the of the mutations by screening for BAPTA and Mn2+ We that was not tolerated at residues with large side chains and were to These were of of residue 783 D. T. Rao R. J. Biol. Cell. 2000; Scholar). mutant V335G a and in to BAPTA and Mn2+ of a change in ion selectivity A was observed for the we of the side at residue 335 could for loss of in the Q783A We that the mutant a in to that of the mutant Q783A In mutations in and also within were in the phenotype of mutant Q783A To these we cation of ATP V335G and were at the with a and purified by acid ATP hydrolysis was as D. T. Rao R. J. Biol. Cell. 2000; and the for Ca2+ and Mn2+ was We have that activity of PMR1 is on the of or ions D. T. Rao R. J. Biol. Cell. 2000; Scholar). Although the affinity for Ca2+ was not different from the V335G mutant an loss in affinity for with the from the phenotype screen we that activity in the Q783A mutant was to was low to D. T. Rao R. J. Biol. Cell. 2000; Scholar). that the for Mn2+ in the mutant was to an effective of the Mn2+ transport Thus, a large reduction in at residue 335 mimics the Q783A an in for the Q783A with a packing interaction between M4 and M6 at these of ATP not activity was low to not activity was low to D. T. Rao R. J. Biol. Cell. 2000; Scholar). in a at 335 and 783 in PMR1 in additional evidence for the interaction between residues 335 and we the of of mutations at these sites by phenotype the of and mutations on growth toxicity of BAPTA and Mn2+ individual mutations that to were for and growth the mutants Q783A and to BAPTA, but the mutant a loss of BAPTA to the Δpmr1 null The was also in with mutations or of these mutations or near phenotype the Q783A and mutant growth in the phenotype In the we observed an by the in with Taken together, results that of side chains at 335 and 783 can either or the of individual of and of and mutations at 783 and 335 in PMR1 at 335 and 783 be Loss of and that the interaction between Gln-783 and Val-335 is it be to the side chains were residues a ion in the could be loss of individual substitutions at these sites to of transport M. A. S. A. 1992; PubMed Scopus Google Scholar). However, of residues in that were to be could not be loss of activity M. A. S. A. 1992; PubMed Scopus Google Scholar). the of phenotype screening of mutants and the We show that has near growth with that substitutions with other residues and were tolerated at this D. T. Rao R. J. Biol. Cell. 2000; Scholar). In mutant an loss of Mn2+ as by a large reduction in to but not to We also an of in this to that of the Δpmr1 null with a loss of Mn2+ transport we show that a at 335 is of loss of Mn2+ to that at that growth sensitivity resulting from the to BAPTA or Mn2+ was not by of the the phenotypic changes were in the The x-ray crystal structure of the SERCA pump at 2.6-Å in the E1 conformation a of membrane helices M5, M6, and that two binding sites for Ca2+ in excellent with from mutagenesis G. D.H. Nature. 1989; PubMed Scopus Google Scholar). of the acid of PMR1 with SERCA an of which to in the regions of M4, M5, and M6 to Ca2+ in SERCA are the residues of SERCA have been by in PMR1 and with this mutagenesis of and residues in transmembrane helices of PMR1 revealed the of residues in ion binding and transport. Thus, substitutions at and in M6 in loss of Ca2+ and Mn2+ transport of in M4 with low of Ca2+ transport A from these is that a cation binding of binding Ca2+ and in PMR1. mutant Q783A at the cytoplasmic interface of M6, loss of Mn2+ Ca2+ transport properties at this not to be as by with and it that Gln-783 Mn2+ the of side chains at this is of a packing with we that substitutions at residue at the cytoplasmic interface of M4, either or the of substitutions at with their from a of M4 and M6 (15Lapinskas P.J. Cunningham K.W. Liu X. Fink G.R. Culotta V. Mol. Cell Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). We of the structural on the and of SERCA C. M. Nomura H. Ogawa H. Nature. 2000; PubMed Scopus Google Scholar, C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google to the of Val-335 and Gln-783 in models of PMR1. the of is on the of we have the to M4, M5, and M6 of PMR1 where with SERCA is high and The of the E1 conformation in a Ca2+ with the residues and essential for from M6 and important for from In the E2 conformation the Ca2+ has been by the of these notably from the binding chains that may be loss of transport activity are in green and can be as from the cation binding A and at two residues in M5, and in loss of this residues into the enclosed by M4, M5, and M6 where they have the to transport The for a or side at residue a in ion is at this in SPCA and but is by a in the SERCA and SPCA are in ion binding within the and it is that to ion binding or transport in these The residue at is in of pumps and must play a in transport. models show a reorientation of the of Phe-738 from a to to the of the Phe-738 is in the of Val-335 and Gln-783 in the E2 structure and may in a packing interaction with these residues The reorientation of the may be important for access of ions from the cytoplasmic Taken together, the of the side chains in are with the results from mutagenesis and the of Val-335 and Gln-783 near the cytoplasmic interface, but not to in the E1 A and in to Thus, Val-335 is by the and from a of Å that is to a interaction between side between in the cytoplasmic of the membrane M4 and M6 of SERCA also be in the E1 crystal structure of Thus, residue and and and and of SERCA form in the E2 conformation D.H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google but are Å in the E1 crystal Importantly, data from these were used to on the of M4 and M6 helices of PMR1 D. T. Rao R. J. Biol. Cell. 2000; which is to be of the E2 in the E2 and Val-335 and Gln-783 in the of and are within of interaction with other and with as by mutagenesis the basis of these we that packing interactions between and Gln-783 at the cytoplasmic interface of membrane helices M4, M5, and M6, Mn2+ transport. the of the acid substitutions and the of these residues to the membrane interface, it is that side chains at 783 or 335 Mn2+ ions. that mutations at these sites can alter access of Mn2+ and Ca2+ to the binding We suggest that the of the side chains of Gln-783 Val-335 and may be critical for Ca2+ and Mn2+ with individual substitutions on these ions. Mn2+ and Ca2+ may the binding pocket different be with that the SPCA are unique in their ability to high affinity Mn2+ to the SERCA and with to Ca2+ transport V.-K. Mandal D. Vahadji C. Rao R. J. Biol. Chem. 2002; 277: 6422-6427Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). is evidence suggesting that residues at the cytoplasmic interface of may form a for Ca2+ in SERCA J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Gln-783 and Val-335 may serve a for Mn2+ in the These may be by of ion binding and in this we that Val-335 and Gln-783 are unique and invariant in known SPCA pumps to their to Mn2+ transport. We for and and for