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Direct Demonstration of Ca2+ Binding Defects in Sarco-Endoplasmic Reticulum Ca2+ ATPase Mutants Overexpressed in COS-1 Cells Transfected with Adenovirus Vectors

1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês

10.1074/jbc.273.24.15104

ISSN

1083-351X

Autores

Christopher Strock, Marco Cavagna, Wendy E. Peiffer, Carlota Sumbilla, David E. Lewis, Giuseppe Inesi,

Tópico(s)

Erythrocyte Function and Pathophysiology

Resumo

Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+inhibition of ATPase phosphorylation by Pi (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 μm Ca2+ and in the absence of ATP, the Glu771 → Gln, Thr799 → Ala, Asp800 → Asn, and Glu908 → Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 → Gln and the Asn796 → Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 → Gln mutant does not bind any Ca2+, and its phosphorylation by Piis not inhibited by Ca2+. On the contrary, the Asn796 → Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 → Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 → Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+can be overcome by raising the Ca2+ concentration to the mm range, consistent with a general effect of mutations on the affinity of the ATPase for Ca2+. Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+inhibition of ATPase phosphorylation by Pi (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 μm Ca2+ and in the absence of ATP, the Glu771 → Gln, Thr799 → Ala, Asp800 → Asn, and Glu908 → Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 → Gln and the Asn796 → Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 → Gln mutant does not bind any Ca2+, and its phosphorylation by Piis not inhibited by Ca2+. On the contrary, the Asn796 → Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 → Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 → Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+can be overcome by raising the Ca2+ concentration to the mm range, consistent with a general effect of mutations on the affinity of the ATPase for Ca2+. Different Effects of Gsα Splice Variants on β2-Adrenoreceptor-mediated Signaling: THE β -ADRENORECEPTOR COUPLED TO THE LONG SPLICE VARIANT OF G α HAS PROPERTIES OF A CONSTITUTIVELY ACTIVE RECEPTORJournal of Biological ChemistryVol. 273Issue 18PreviewThe β2-adrenoreceptor (β2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsα differ by a 15-amino acid insert between the Ras-like domain and the α-helical domain. The long splice variant of Gsα (GsαL) binds GDP with lower affinity than the short splice variant (GsαS), but the impact of this difference on the interaction of Gsα with the β2AR is not known. We studied the β2AR/Gsα interaction using receptor/G-protein fusion proteins (β2ARGsαS and β2ARGsαL) expressed in Sf9 cells. Full-Text PDF Open AccessDifferent effects of Gsα splice variants on β2-adrenoreceptor-mediated signaling. The β2-adrenoreceptor coupled to the long splice variant of Gsα has properties of a constitutively active receptor.Journal of Biological ChemistryVol. 273Issue 18PreviewDue to a printer's error in various places throughout the article mg was incorrectly printed as μg and in a few places mCi and mm were printed instead of μCi and μm. The article is printed in its entirety on the following pages, using original page numbers. Full-Text PDF Open Access Activation of the sarco-endoplasmic reticulum Ca2+ (SERCA) 1The abbreviations used are: SERCA, sarco-endoplasmic reticulum Ca2+; SR, sarcoplasmic reticulum; Cr-ATP, chromium ATP; EGFP, enhanced green fluorescence protein; pfu, plaque-forming unit; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; Mes, 4-morpholineethanesulfonic acid. ATPase requires binding of Ca2+, which is then moved across the membrane upon utilization of ATP. Equilibrium binding isotherms, obtained with sarcoplasmic reticulum (SR) vesicles as an abundant source of enzyme, demonstrate that in the absence of ATP, two Ca2+/ATPase bind cooperatively with K a = 5 × 1012m−2 (2Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). As for the topology of Ca2+ binding within the ATPase molecule, involvement of six amino acid residues (Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908) within four clustered transmembrane helices (M4, M5, M6, and M8) was suggested by mutational analysis (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar). The low yield of recombinant enzymes, however, has limited direct measurements of Ca2+ binding to the pertinent mutants (with the exception of the Glu309 → Gln mutant; Skerjank et al. (3Skerjanc I.S. Toyofuku T. Richardson C. MacLennan D.H. J. Biol. Chem. 1993; 268: 15944-15950Abstract Full Text PDF PubMed Google Scholar)). In fact, involvement of the six amino acids in Ca2+ binding was suggested by their mutations interfering with the inhibitory effect of Ca2+ on enzyme phosphorylation by Pi. The shortcoming of such experiments, however, was that they did not distinguish direct effects of mutations on Ca2+ binding from mutational effects on transmission of the Ca2+ binding signal within the ATPase protein. Alternative studies were performed to test whether the pertinent mutants retain the ability to occlude Ca2+ following addition of Cr-ATP (4Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar). In this case, however, there is some degree of uncertainty as to whether Cr-ATP may affect Ca2+ binding by stabilizing the enzyme in a state analogous to that of the phosphorylated intermediate (i.e. "E2"). Other studies have utilized the effect of Ca2+ on the susceptibility of wild type and mutated enzyme to proteolytic digestion (5Falson P. Menguy T. Corre F. Bouneau L. De Gracia A.G. Soulié S. Centeno F. Moller J.V. Champeil P. Le Maire M. J. Biol. Chem. 1997; 272: 17258-17262Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We have now improved the efficiency of ATPase gene transfer into COS-1 cells by using recombinant adenovirus vectors. The resulting expression yields sufficient amounts of protein for direct measurements of Ca2+ binding in the absence of nucleotide substrate, using native microsomal vesicles as the source of enzyme. We describe here our recombinant adenovirus constructs, the characteristics of ATPase overexpression in COS-1 cells under control of the SV40 promoter, the direct measurements of Ca2+ binding by wild type and mutated enzyme, and related tests of Ca2+ inhibition of the enzyme reaction with Pi. The chicken fast muscle SR (SERCA-1) ATPase cDNA (6Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (74) Google Scholar) was inserted into the pUC19 plasmid for amplification and then subcloned into the pSELECT-1 vector for site-directed mutagenesis by the Altered Sites In Vitro Mutagenesis System made available by Promega (Madison, WI) or by overlap extension using polymerase chain reaction (7Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6963) Google Scholar). Eleven unique restriction sites were introduced into the SERCA-1 cDNA, retaining the original coding sequence. These sites are spaced at approximately equal intervals and facilitate further mutagenesis by generating cassettes of approximately three hundred bases that can be conveniently sequenced following mutagenesis and exchanged with the corresponding cassette in the wild type cDNA. Furthermore a c-myc tag was added to the 3′ end to monitor ATPase expression using anti-c-myc antibodies, independently of mutations in the enzyme sequence. Site-directed mutations were produced in the SERCA-1 cDNA by using the Altered Sites In Vitro Mutagenesis System (Promega) or by overlap extension using the polymerase chain reaction (7Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6963) Google Scholar). Enhanced green fluorescence protein (EGFP) cDNA (containing the Phe64 → Leu and Ser65 → Thr mutations of wild type green fluorescence protein) was obtained fromCLONTECH (Palo Alto) in the form of the pEGFP-1 plasmid. Wild type and mutated cDNA, as well as the EGFP cDNA, was subcloned into the shuttle plasmid pΔE1sp1A (Microbix BioSystems). In the final constructs, the cDNA was preceded by the SV40 promoter (cytomegalovirus promoter in the case of EGFP) and followed by the SV40 polyadenylation signal, both obtained from the mammalian expression plasmid pCDL-SRα296 (8Takebe Y. Seiki M. Fujisawa J.I. Hoy P. Yokota K. Arai K.I. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). The shuttle plasmid was either used directly for transfection of COS-1 cells by the DEAE-dextran method or alternatively for cotransfection of HEK293 cells in conjunction with the replication defective adenovirus plasmid pJM17 (Microbix BioSystems) to obtain recombinant adenovirus vectors (9Graham F.L. Prevec L. Vaccines: New Approaches to Immunological Problems. Butterworth-Heinemann, Boston, MA1992: 363-390Crossref Google Scholar). The shuttle vector was constructed such that homologous recombination would result in an antisense direction of the SERCA cDNA with respect to the original adenovirus E1 gene and its promoter. The recombinant products were plaque-purified and cesium-banded, yielding concentrations on the order of 1010 pfu/ml. Analogous procedures were used to obtain recombinant EGFP adenovirus vectors. Cultures of HEK293 and COS-1 cells were maintained as described by Graham and Prevec (9Graham F.L. Prevec L. Vaccines: New Approaches to Immunological Problems. Butterworth-Heinemann, Boston, MA1992: 363-390Crossref Google Scholar) and Sumbilla et al. (10Sumbilla C. Lu L. Lewis D.E. Inesi G. Ishii T. Takeyasu K. Fang Y. Fambrough D.M. J. Biol. Chem. 1993; 268: 21185-21192Abstract Full Text PDF PubMed Google Scholar), respectively. The growth medium for COS-1 cells was Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and containing Penn-Strep (100 units/ml) and Fungizone (1 μg/ml). Transfections of COS-1 cells with wild type or mutated SERCA-1 cDNA or EGFP cDNA subcloned into the shuttle vector pΔE1sp1A as described above were conducted by the DEAE-dextran method as described by Sumbilla et al. (10Sumbilla C. Lu L. Lewis D.E. Inesi G. Ishii T. Takeyasu K. Fang Y. Fambrough D.M. J. Biol. Chem. 1993; 268: 21185-21192Abstract Full Text PDF PubMed Google Scholar). Recombinant adenovirus vectors were used as follows: 6- or 15-cm plates of COS-1 cells (75–80% confluency) were aspirated to remove growth medium and then layered with 1 or 5 ml of infection medium (Dulbecco's modified Eagle's medium supplemented with 5% horse serum, 100 units Penn-Strep/ml, and 1 μg Fungizone/ml) containing 1.4 × 108 pfu/ml corresponding to about 100 pfu/cell. 1 h after infection, 4 or 20 ml of growth medium was added, and 2 days later the cells were harvested for fluorescence microscopy with or without immunostaining and for microsomal preparations. Lawns of cultured cells were first washed with PBS and then fixed with 4% formaldehyde for 10 min. After repeated washings with PBS, blocking of nonspecific sites was obtained by a 10-min incubation with 1% bovine serum albumin and 0.5% lysine in PBS. This was followed by a 45-min incubation with the primary antibody at a concentration of 5–10 μg/ml of PBS containing 1% albumin, 0.5% lysine, and 0.25% saponin (permeabilization medium). After washing with PBS, the cells were incubated for 45 min with biotinylated anti-mouse secondary antibody (Vector Laboratory, Burlingame, CA) at a concentration of 5 μg/ml of permeabilization medium. The cells were then washed with PBS and incubated for 20 min with Fluorescein Streptavidin (Amersham Pharmacia Biotech) at a concentration of 5 μg/ml of permeabilization medium. The sample was then washed again with PBS, 70% EtOH, and 90% EtOH, allowed to dry, and processed for fluorescence microscopy using a Zeiss Axioskop equipped with a mercury vapor lamp and fluorescence accessories. The procedure for microsome preparation was as described by Autry and Jones (11Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), and the final product was stored in small aliquots at −70 °C. The total microsomal protein was determined using bicinchoninic acid assay (Pierce). The expressed SERCA-1 ATPase was detected by Western blotting. For this purpose, microsomal protein were separated in 7.5% Laemmli (12Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212371) Google Scholar) electrophoretic gels blotted onto nitrocellulose paper. This was then incubated with a monoclonal antibody (CaF3–5C3) to the chicken SERCA-1 (6Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (74) Google Scholar) and in parallel with a monoclonal antibody (9E10) to the c-myc epitope (13Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2221) Google Scholar). After incubation with secondary antibody (goat anti-mouse IgG-horseradish peroxidase-conjugated), the bound proteins were probed using an Enhanced Chemiluminescence-linked detection system (Amersham Pharmacia Biotech). Quantitation of immunoreactivity was obtained by densitometry and standardized with samples of the wild type ATPase used as controls for the functional studies. Studies of Ca2+ transport and phosphoenzyme formation with [32P]phosphate were carried out as described by Inesi et al. (14Inesi G. Kurzmack M. Lewis D. Methods Enzymol. 1988; 157: 154-190Crossref PubMed Scopus (56) Google Scholar) and detected by autoradiography. ATPase hydrolytic activity was assessed by measuring Pi production (15Lanzetta P.A. Alvarez L.J. Reinsch P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1859) Google Scholar). Ca2+ binding to the expressed ATPase in the absence of ATP was measured by a filtration method. The equilibration mixture contained 20 mm MOPS, pH 6.8, 80 mm KCl, 3 mm MgCl2, 20 μm[45Ca]CaCl2 (including endogenous Ca2+), and 400 μg microsomal protein/ml. Endogenous Ca2+ in the equilibration medium was determined by titrating with EGTA in the presence of 50 μm Arsenazo and recording differential light absorption changes (660 and 687 nm wavelengths) with a DW-2000 SLM-Aminco spectrophotometer. Controls for nonspecific binding were conducted with microsomes preincubated with thapsigargin (6.75 μg/mg protein), and the binding observed with the inhibited microsomes was subtracted from the total binding obtained with noninhibited microsomes to yield "specific" Ca2+ binding. The Ca2+ binding assay was started by adding either 2.7 μg of thapsigargin in 2 μl of Me2SO or 2 μl of Me2SO alone to approximately 400 μg of microsomal protein in 50 μl of medium. After a brief incubation at room temperature, the samples were kept on ice for 10 min, and then 1.0 ml of binding medium was added. Following a 10-min incubation on ice, 0.75 ml was placed on a Millipore filter (HAWP 0.65 μm, 25-mm diameter) under suction for approximately 30 s. The vacuum was then turned off, and the filter was blotted on a paper towel to remove excess moisture, folded, and inserted into a 7-ml scintillation vial. The filters were dissolved with 1 ml of N ,N -dimethylformamide, scintillation fluid was added, and the radioactivity was measured by scintillation counting. The measured Ca2+ binding levels were finally adjusted to compensate for slight variations of SERCA-1 expression in various preparations, with reference to a wild type preparation as indicated by Western blots. The thapsigargin-sensitive (i.e. specific) binding accounted for approximately 25% of the total Ca2+ binding by microsomes obtained from cells expressing wild type SERCA. It is known that thapsigargin is a global SERCA inhibitor (16Inesi G. Sagara Y. Arch. Biochem. Biophys. 1992; 298: 313-317Crossref PubMed Scopus (163) Google Scholar), and we established that at the concentration used thapsigargin inhibited the Pi reaction in the wild type enzyme and in all mutants studied. A preliminary assessment of adenovirus vector efficiency in COS-1 cells was obtained by microscopic visualization of the number of COS-1 cells expressing EGFP. Fig. 1 (A andB ) shows phase contrast and fluorescent images of COS-1 cells following the DEAE-dextran transfection procedure. A comparison of the two images shows that only a few cells express EGFP. On the other hand, in Fig. 1 C , nearly all cells express EGFP following infection by recombinant EGFP adenovirus (40 pfu/cell). A quantitative assessment of the percentage of cells expressing EGFP as a function of adenovirus concentration is given in Fig.2. An asymptotic level of 97–100% transfection efficiency was obtained at 100 pfu/seeded cell, although the amount of expressed protein continued to rise as the number of gene copies introduced per cell was further increased.Figure 2Gene transfer efficiency as a function of recombinant adenovirus pfu level. COS-1 cells were infected with increasing recombinant adenovirus pfu/cell ratios as described under "Materials and Methods." The EGFP cDNA was under control of cytomegalovirus promoter. 2 days following the infection, 10 different fields of each culture plate were examined by comparative phase and fluorescence microscopy to determine the percentage of cells expressing EGFP. The cells were then scraped off the plates, suspended in 50 mm NaH2PO4, 10 mm Tris, pH 8.0, and 200 mm NaCl, and lyzed by several passes through a 22 gauge syringe needle. Fluorescence intensity was then measured at 488 nm excitation wavelength and 510 nm emission wavelength. Note that as the pfu level increases the percentage of fluorescent cells begins to level off as the 100% asymptote is reached. On the other hand, the fluorescence intensity continues to rise likely because of a rise of the virus copy number in each cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ATP-dependent Ca2+ transport is a highly specific functional parameter of SERCA that can be conveniently measured by following the uptake of radioactive calcium tracer by microsomal vesicles. Microsomes obtained from control COS-1 cells do not exhibit a significant rate of Ca2+ transport (Fig.3). On the other hand, we found that under optimal conditions, the rate of Ca2+ transport by microsomes obtained from cells infected with the SERCA-1 adenovirus vector (100 pfu/cell) was approximately 10-fold higher than that of microsomes obtained from cells transfected by the DEAE-dextran method (Fig. 3). Conversely, their transport activity was approximately 10-fold lower than the rate of Ca2+ transport by native SR vesicles obtained from rabbit skeletal muscle (data not shown). Because SERCA-1 accounts for approximately 50% of the total protein in SR vesicles, it is apparent that transgenic SERCA-1 accounts for approximately 5.0% of the total microsomal protein obtained from COS-1 cells infected with recombinant adenovirus under our conditions. Similar conclusions were reached by comparative measurements of Ca2+-dependent ATPase activity (not shown). They are also consistent with Western blots showing no SERCA in control samples (Fig. 3, inset , lane 1 ) and much greater amounts of recombinant SERCA in microsomes obtained from cells infected with adenovirus (Fig. 3, inset , lanes 4 and5 ) as compared with microsomes obtained from cells transfected by the DEAE-dextran method (Fig. 3, inset ,lanes 2 and 3 ). Ca2+ binding in the absence of ATP may be considered as the initial step of a single catalytic and transport cycle. In fact it is well known that Ca2+ binding to the ATPase is required to activate the enzyme before ATP can be utilized. The bound calcium is then displaced vectorially and released against a concentration gradient upon enzyme phosphorylation by ATP. The cycle is finally completed by hydrolytic cleavage of the phosphorylated enzyme intermediate. We have previously used chromatography equilibration columns to measure Ca2+ binding to SR ATPase under equilibrium conditions, in the absence of ATP. The resulting binding isotherms demonstrate that two Ca2+ bind cooperatively to each ATPase withK a = 5 × 1012m−2 at neutral pH (2Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). This method, however, is not suited for measurements of Ca2+ binding to COS-1 microsomes because of the low yield of recombinant enzyme. For this reason we used the filtration method, which has the advantage of being suited for small protein samples, even though it is less than optimal from an equilibrium theory viewpoint. Prior to its use, we determined the linear range for protein collection by Millipore filters using SR microsomal protein as a standard. It is shown in Fig.4 that the linear range extended to 500 μg of microsomal protein/filter. We found that 20 μmCa2+ provided an optimal ligand concentration for saturating the high affinity sites, yielding binding data consistent with those obtained by column chromatography (2Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar). We also found that inhibition with thapsigargin provided a very useful tool to demonstrate the specificity of the high affinity Ca2+ binding to the SERCA enzyme. Therefore, the data reported here correspond to the difference between Ca2+ binding in the absence and in the presence of thapsigargin. Using the filtration method under these conditions, we obtained Ca2+ binding levels of 7.0 nmol/mg of SR protein and 0.7 nmol/mg of microsomal protein derived from COS-1 cells infected with SERCA adenovirus vector. These values are consistent with the 10-fold difference observed in the Ca2+ transport and ATPase activities of the two preparations. No significant Ca2+binding signal was detected with microsomes derived from COS-1 cells transfected by the DEAE-dextran method, because of the low SERCA content of these microsomes. We observed in our Western blots only slight variations in the levels of SERCA-1 protein recovered in the microsomal fraction of COS-1 cells expressing the various SERCA mutants under the same conditions (Fig.5). We obtained densitometric evaluations of the electrophoretic bands to relate the functional parameters measured for each mutant to a corresponding quantity of wild type SERCA-1. We found, however, that the Glu309 → Gln, Asp771 → Asn, Asn796 → Ala, Thr799 → Ala, Asp800 → Asn, and Glu908 → Ala mutants do not sustain significant rates of either ATP hydrolysis or coupled Ca2+ transport (see also Clarke et al. (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar)). Furthermore, formation of phosphorylated intermediate through Ca2+-dependent ATP utilization is totally inhibited in these mutants. On the other hand, phosphoenzyme formation by utilization of Pi still occurs at normal levels (see below). The main aim of our studies was to measure Ca2+ binding by the ATPase mutants, taking advantage of their overexpression in COS-1 cells infected with the adenovirus vectors. In fact, we were able to unambiguously demonstrate specific defects of Ca2+ binding in these mutants. It is shown in Table I that at neutral pH and in the presence of 20 μm Ca2+, some of the mutants (i.e. Glu771 → Gln, Thr799 → Ala, Asp800 → Asn, and Glu908 → Ala) displayed no significant binding, whereas others (i.e. Glu309 → Gln and Asn796 → Ala) bind approximately half as much Ca2+ as the wild type enzyme (Fig. 6). A 50% reduction of Ca2+ binding by the Glu309 → Gln mutation was also noted by Skerjanc et al. (3Skerjanc I.S. Toyofuku T. Richardson C. MacLennan D.H. J. Biol. Chem. 1993; 268: 15944-15950Abstract Full Text PDF PubMed Google Scholar). Interestingly, we found that if the pH is reduced to 6.2, Ca2+ binding by the Glu309 → Gln mutant becomes negligible (0.02 ± 0.24 nmol/mg protein), whereas the Asn796 → Ala mutant binds approximately the same level of Ca2+ (0.44 ± 0.39 nmol/mg protein) as at neutral pH.Table IATP-independent Ca2+ binding by wild type and mutant SERCA-1Calcium boundn1n2nmolWild type0.70 ± 0.311256Glu309 → Gln0.30 ± 0.158635Asp800 → Asn0.03 ± 0.13419Glu908 → Ala−0.033 ± 0.15526Glu771 → Gln−0.01 ± 0.17525Asn796 → Ala0.27 ± 0.191042Thr799 → Ala−0.03 ± 0.10420Ca2+ binding was measured at pH 7.0, as described under "Materials and Methods," and is reported in nmol/mg of microsomal protein ± S.D. n1 is the number of experiments performed for each wild type or mutant SERCA, and n2 is the total number of binding measurements (i.e. 4–6 binding measurements/experiment). Note that the level of Ca2+ binding by the Glu309 → Gln and Asn796 → Ala mutants is approximately half as much as that by the wild type SERCA-1. Binding by the other mutants is negligible. Open table in a new tab Ca2+ binding was measured at pH 7.0, as described under "Materials and Methods," and is reported in nmol/mg of microsomal protein ± S.D. n1 is the number of experiments performed for each wild type or mutant SERCA, and n2 is the total number of binding measurements (i.e. 4–6 binding measurements/experiment). Note that the level of Ca2+ binding by the Glu309 → Gln and Asn796 → Ala mutants is approximately half as much as that by the wild type SERCA-1. Binding by the other mutants is negligible. As mentioned above, putative Ca2+ binding defects of these mutants were originally suggested by the lack of inhibition of the Pi reaction by Ca2+ (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar). With our present experiments we first confirmed that the Pi reaction is in fact inhibited by 20 μm Ca2+, at pH 7.0 or 6.2 when the wild type ATPase is used and is not inhibited when the Glu771 → Gln, Thr799 → Ala, Asp800 → Asn, and Glu908 → Ala mutants are used (Fig. 7). We also found that the Pi reaction is inhibited by 20 μmCa2+ at pH 7.0 or 6.2 when the Asn796 → Ala mutant is used, similarly to the wild type ATPase. On the other had, when the Glu309 → Gln is used, the Pireaction is inhibited by 20 μm Ca2+ at pH 7.0 but not at pH 6.2 (Fig. 7; see also Ref. 17Andersen J.P. Vilsen B. FEBS Lett. 1992; 306: 247-250Crossref PubMed Scopus (24) Google Scholar). With all mutants inhibition of the Pi reaction was obtained if the Ca2+ concentration was raised to the mm level (not shown, but see Ref. 18Chen L. Sumbilla C. Lewis D. Zhong L. Strock C. Kirtley M.E. Inesi G. J. Biol. Chem. 1996; 271: 10745-10752Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Identical results were obtained when the experiments were performed in the presence of the ionophore A23187, which increases the membrane permeability to Ca2+ and allows ATPase exposure to Ca2+ from both sides of the membrane. Equilibrium and kinetic experiments have demonstrated that in the absence of ATP, SERCA binds Ca2+ with a stoichiometry of two Ca2+/enzyme. Binding occurs with a sequential and cooperative mechanism (2Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar, 19Dupont Y. Biochim. Biophys. Acta. 1982; 688: 75-87Crossref PubMed Scopus (101) Google Scholar, 20Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) whereby the enzyme is stabilized in the "E1" state. An important discovery for the understanding of the Ca2+ binding topology within the SERCA molecule was achieved when single mutations of specific residues in the transmembrane domain were found to interfere with Ca2+inhibition of enzyme phosphorylation by Pi (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar). Based on this interference, it was proposed that the six residues participate in Ca2+ complexation within the transmembrane domain (1Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (505) Google Scholar). In fact it was shown by molecular modeling (21Inesi G. Lewis D. Nikic D. Hussain A. Kirtley M.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 185-215PubMed Google Scholar) that within the constraints imposed by the positions of these residues on four transmembrane helices, it is possible to arrange their oxygen functions to form a duplex calcium binding site with distribution and distances compatible with other duplex sites of known crystal structure, such as that of thermolysin (22Holmes M.A. Matthews B.W. J. Mol. Biol. 1982; 160: 623-639Crossref PubMed Scopus (448) Google Scholar). Several attempts have been made to assign the oxygen functions of the six amino acids implicated in binding to either calcium ion of the duplex complex (21Inesi G. Lewis D. Nikic D. Hussain A. Kirtley M.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 185-215PubMed Google Scholar, 23Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (118) Google Scholar, 24Rice W.J. Green N.M. MacLennan D.H. J. Biol. Chem. 1997; 272: 31412-31419Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Additional amino acids within a neighboring cytosolic segment have been also implicated (5Falson P. Menguy T. Corre F. Bouneau L. De Gracia A.G. Soulié S. Centeno F. Moller J.V. Champeil P. Le Maire M. J. Biol. Chem. 1997; 272: 17258-17262Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, a shortcoming of this work has been the lack of direct Ca2+binding measurements with the mutants, because of the low quantities of recombinant protein. The only mutant tested for binding was Glu309 → Gln expressed in insect cells infected with recombinant baculovirus (3Skerjanc I.S. Toyofuku T. Richardson C. MacLennan D.H. J. Biol. Chem. 1993; 268: 15944-15950Abstract Full Text PDF PubMed Google Scholar). This measurement required solubilization of the membrane-bound enzyme, purification by affinity chromatography, and reconstitution in liposomes, leaving some uncertainty on the detergent effect on binding and the orientation of the reconstituted enzyme in the liposomal membrane. Alternatively, "Ca2+occlusion" was measured in the presence of Cr-ATP (4Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar), which stabilizes the enzyme in a state similar to that obtained by enzyme phosphorylation with ATP. An advantage of our present experiments is that Ca2+ binding was measured in the absence of ATP under equilibrium conditions yielding strictly the E1 state. Another advantage is the use of wild type and mutant proteins assembled in native microsomal membrane with no need for detergent solubilization. Our measurements demonstrate that the Glu771 → Gln, Asp800 → Asn, Thr799 → Ala, and Glu908 → Ala mutations result in total loss of Ca2+ binding in the presence of 20 μmCa2+ (Table I). The strong inhibition of Ca2+binding in the first two mutants may be explained by considering that Glu771 and Asp800 could contribute electronegativity to both calcium ions, each donating its carboxyl oxygen to one calcium and its carbonyl oxygen to the other. Yet, it is remarkable that mutation of a single one of the acidic amino acids to its corresponding amide (thereby leaving the carbonyl oxygen in place) results in interference with binding of both calcium ions and with inhibition of the Pi reaction by Ca2+ (TableI). The strong inhibition of Ca2+ binding by the Thr799 → Ala and Glu908 → Ala mutations is also remarkable, because Thr can contribute only one hydroxyl group to the coordination complex. Furthermore, Glu908 appears to contribute only a carbonyl group because the Glu908 → Gln mutation does not have functional consequences (18Chen L. Sumbilla C. Lewis D. Zhong L. Strock C. Kirtley M.E. Inesi G. J. Biol. Chem. 1996; 271: 10745-10752Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Clarke D.M. Loo T.W. MacLennan D.H. J. Biol. Chem. 1990; 265: 6262-6267Abstract Full Text PDF PubMed Google Scholar). It is then apparent that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 provide important stabilization to the cooperative Ca2+ complex, either by direct interaction with Ca2+ or by participation in hydrogen bonding with water or peptide amide functions, so much so that mutational interference with a single one of these functions results in major disruption of the duplex binding site. These effects, however, can be overcome by increasing the Ca2+concentration (18Chen L. Sumbilla C. Lewis D. Zhong L. Strock C. Kirtley M.E. Inesi G. J. Biol. Chem. 1996; 271: 10745-10752Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), indicating that mutational disruption affects the affinity of the enzyme for Ca2+. It is noteworthy that our finding of strong binding inhibition in the Glu908 → Ala mutant in the absence of ATP (i.e. E1 state) is in apparent contrast with the Ca2+ occlusion by the same mutant in the presence of Cr-ATP (4Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar, 23Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (118) Google Scholar). This may be explained by the high concentration of Ca2+ used in these experiments (23Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (118) Google Scholar). Although we observed strong inhibition of Ca2+ binding with the mutants mentioned above we found that in the presence of 20 μm Ca2+ and pH 7.0, the Glu309→ Gln and Asn796 → Ala mutations result in reduction of Ca2+ binding to approximately half the level observed with wild type enzyme (Fig. 6). At pH 6.2, on the other hand, the Glu309 → Gln mutant exhibits no significant Ca2+ binding, whereas the Asn796 → Ala mutant retains the same binding as at neutral pH. This suggests that ionization of acidic functions of other amino acids (e.g. Glu771 or Asp800) occurs as the pH is shifted from 6.2 to 7.0, thereby facilitating Ca2+ binding in the Glu309 → Gln mutant. On the other hand, in the Asn796 → Ala mutant, the presence of the Glu309 acidic function allows binding of inhibitory Ca2+ even at pH 6.2. The lack of Ca2+ inhibition of the Pi reaction in the mutants allowing no Ca2+ binding is well understandable. On the other hand, the strong inhibition of the Pi reaction in mutants permitting binding of half the normal Ca2+ level suggests that (a ) these mutant molecules bind only one of two Ca2+ known to bind to the wild type enzyme and (b ) the single bound Ca2+is sufficient to inhibit the Pi reaction. Experimental demonstration of the inhibition of the Pi reaction by single Ca2+ binding is quite satisfactory, because it is observed in both Asn796 → Ala and Glu309 → Gln mutants at pH 7.0 and only in the former mutant at pH 6.2. That a single Ca2+ may be sufficient to inhibit the Pi reaction was previously suggested based upon the effects of sequential (and negatively cooperative) binding of strontium to sarcoplasmic reticulum ATPase (26Fujimori T. Jencks W.P. J. Biol. Chem. 1992; 267: 18475-18487Abstract Full Text PDF PubMed Google Scholar) and on different effects of various mutations on the inhibition of the Pi reaction by Ca2+ (23Andersen J.P. Biosci. Rep. 1995; 15: 243-261Crossref PubMed Scopus (118) Google Scholar). Our direct measurements of Ca2+binding at pH 7.0 and 6.2 in parallel with phosphorylation experiments confirm that in the Asn796 → Ala and Glu309→ Gln mutants, at suitable Ca2+ and H+concentrations, a single Ca2+ is sufficient to inhibit the Pi reaction. This would be unlikely to occur in the wild type enzyme, because of the highly cooperative character of Ca2+ binding.

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