Role of the S3 Stalk Segment in the Thapsigargin Concentration Dependence of Sarco-endoplasmic Reticulum Ca2+ ATPase Inhibition

The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is specifically inhibited by thapsigargin (TG), whereas the Na+,K+-ATPase is not. Large chimeric exchanges between Ca2+ and Na+,K+-ATPases (Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem.269, 26598–26601), as well as photolabeling with a TG azido derivative (Hua, S., and Inesi, G. (1997) Biochemistry36, 11865–11872), suggest that the S3-M3 (stalk and membrane-bound) region of the Ca2+ ATPase is involved in TG binding. We produced small site-directed changes in the S3 stalk segment of the Ca2+ ATPase and found that mutation of five amino acids to the corresponding Na+,K+-ATPase residues increases by 3 orders of magnitude the TG concentration required for inhibition of Ca2+ ATPase and coupled Ca2+transport. A single mutation in the S3 stalk segment (Gly257 → Ile) is sufficient to increase by 1 order of magnitude the TG concentration required to produce 50% inhibition. By comparison, mutations yielding a nine-amino acid homology in the M3 transmembrane segment, or a 25-amino acid homology in the S4 stalk segment, do not affect the ATPase sensitivity to TG. We suggest that specific binding of TG to the S3 stalk segment, in addition to stacking of the TG ring structure at the membrane interface, determines the high affinity of the ATPase for the inhibitor. The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is specifically inhibited by thapsigargin (TG), whereas the Na+,K+-ATPase is not. Large chimeric exchanges between Ca2+ and Na+,K+-ATPases (Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem.269, 26598–26601), as well as photolabeling with a TG azido derivative (Hua, S., and Inesi, G. (1997) Biochemistry36, 11865–11872), suggest that the S3-M3 (stalk and membrane-bound) region of the Ca2+ ATPase is involved in TG binding. We produced small site-directed changes in the S3 stalk segment of the Ca2+ ATPase and found that mutation of five amino acids to the corresponding Na+,K+-ATPase residues increases by 3 orders of magnitude the TG concentration required for inhibition of Ca2+ ATPase and coupled Ca2+transport. A single mutation in the S3 stalk segment (Gly257 → Ile) is sufficient to increase by 1 order of magnitude the TG concentration required to produce 50% inhibition. By comparison, mutations yielding a nine-amino acid homology in the M3 transmembrane segment, or a 25-amino acid homology in the S4 stalk segment, do not affect the ATPase sensitivity to TG. We suggest that specific binding of TG to the S3 stalk segment, in addition to stacking of the TG ring structure at the membrane interface, determines the high affinity of the ATPase for the inhibitor. The sesquiterpene lactone thapsigargin (TG), 1The abbreviations used are: TG, thapsigargin; MOPS, 3-(N-morpholino)propanesulfonic acid; PCR, polymerase chain reaction; bp, base pair(s); SERCA, sarco-endoplasmic reticulum Ca2+ ATPase. isolated from the plant Thapsia garganica (3Christensen S.B. Acta Chem. Scand. 1988; B42: 623-628Crossref Google Scholar), is a highly specific inhibitor of sarco-endoplasmic reticulum Ca2+ ATPases (SERCAs) (4Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3009) Google Scholar,5Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar). Kinetic studies indicate that a 1:1 TG-ATPase stoichiometric complex is formed at extremely low concentration of TG (6Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar). The inhibition involves Ca2+ binding, formation of phosphorylated intermediate, ATP hydrolysis, and Ca2+transport (7Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar, 8Kijima Y. Ogunbunmi E. Fleischer S. J. Biol. Chem. 1991; 266: 22912-22918Abstract Full Text PDF PubMed Google Scholar, 9Witcome M. Henderson I. Lee A.G. East J.M. Biochem. J. 1992; 283: 525-529Crossref PubMed Scopus (80) Google Scholar, 10De Jesus F. Girardet J.-L. Dupont Y. FEBS Lett. 1993; 332: 229-232Crossref PubMed Scopus (28) Google Scholar). These effects suggest a global effect of TG on the enzyme, as also indicated by its influence on formation of ordered ATPase arrays (11Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar, 12Stokes D.L. Lacapére J.-J. J. Biol. Chem. 1994; 269: 11606-11613Abstract Full Text PDF PubMed Google Scholar). Hence, clarification of the TG binding domain would contribute to the understanding of structural and mechanistic features in the enzyme. A useful approach, in this regard, is to study the TG sensitivity of chimeric proteins consisting of defined parts of SERCA and Na+,K+-ATPase, because TG interacts specifically with the former and not with the latter. Previous studies with large chimeric exchanges, however, produced strong inhibition of catalytic turnover and transport. Nevertheless, Ca2+-dependent formation of phosphorylated intermediate was preserved, and a reduced sensitivity of this parameter to TG was obtained upon chimerization of the entire S3-M3 (stalk and membrane-bound) region (13Norregaard A. Vilsen B. Andersen J.P. FEBS Lett. 1993; 336: 248-254Crossref PubMed Scopus (16) Google Scholar). In contrast, the TG sensitivity was not altered significantly if other large regions were exchanged (13Norregaard A. Vilsen B. Andersen J.P. FEBS Lett. 1993; 336: 248-254Crossref PubMed Scopus (16) Google Scholar, 14Ishii T. Takayasu K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8881-8885Crossref PubMed Scopus (33) Google Scholar, 15Sumbilla 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). We describe here the construction and functional characterization of more discrete chimeric changes, involving stepwise mutations of one and up to several amino acids in the S3 or S4 stalk segments of the SERCA, to match the corresponding residues of the Na+,K+-ATPase. A similar strategy was previously used in studies of ouabain binding by the Na+,K+-ATPase, taking advantage of ouabain-sensitive and ouabain-insensitive isoforms of the enzyme (16Lingrel J.B. Arguello J.M. Van Huysse J. Kuntzweiler T.A. Ann. N. Y. Acad. Sci. 1997; 834: 194-206Crossref PubMed Scopus (79) Google Scholar). The chicken fast muscle SERCA-1 cDNA (17Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar), containing 11 artificial and unique restriction sites spaced at approximately 300-bp intervals to facilitate cassette exchange (18Zhang Z. Sumbilla C. Lewis D. Summers S. Klein M.G. Inesi G. J. Biol. Chem. 1995; 270: 16283-16290Abstract Full Text Full Text PDF PubMed Google Scholar), was subcloned into pUC19 vector for site-directed mutagenesis. For this purpose, the cassette delimited byBssHII and Bsu36I restriction sites (579 bp including S3 and M3 coding sequences), and the cassette delimited byBamHI and Bsu36I (318 bp including M4 and S4 coding sequences), were amplified by PCR using oligonucleotide “flanking” primers. Furthermore, complementary mutagenic oligonucleotides of 23–35 bp length were synthesized for each individual mutation. These primers were utilized to hybridize DNA sequences internal to the flanking primers and were used for PCR mutagenesis by the overlap extension method as described by Ho et al. (19Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 51-59Crossref PubMed Scopus (6850) Google Scholar). Briefly, two overlapping fragments containing the mismatched base(s) of the targeted sequence were amplified in separate PCR reactions. The PCR mixtures contained 1 μm each of flanking and mutagenic primers, 800 μm dNTPs, 20 ng of SERCA-1 cDNA, 2.5 units of Pfu (Pyrococcus furiosus) DNA polymerase, and Pfu buffer (Stratagene, Menasha, WI) in a final volume of 100 μl. The reaction products were separated by electrophoresis on a 3% low melting agarose gel (FMC, Rockland, ME), and the appropriate M r band was excised and melted at 72 °C for 5 min. The eluted fragments were fused, and the entire cassette was amplified using both flanking primers. The mutant cassette was then exchanged with the corresponding cassette of wild-type cDNA in pUC19 vector, and sequenced by the dideoxy chain-termination method using Sequenase (U. S. Biochemical Corp.). Additive chimeric mutations were introduced by sequential PCR mutagenesis using mutant DNA as template. Finally, the mutated cDNA was subcloned into COS-1 expression vector pCDL-SRα296 (20Takebe 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) for transfection and overexpression of protein under control of the SV40 promoter. COS-1 cell cultures and transfections were carried out as described by Sumbilla et al. (15Sumbilla 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). The microsomal fraction of transfected COS-1 cells was obtained by differential centrifugation of homogenized cells (15Sumbilla 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). Immunodetection of expressed ATPase in the microsomal fraction was obtained by Western blotting, using the CaF-5C3 monoclonal antibody to SERCA-1 (17Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar), as described by Sumbilla et al. (15Sumbilla 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). ATP-dependent Ca2+ transport was measured by following the uptake of radioactive calcium tracer by microsomal vesicles. The reaction mixture contained 20 mm MOPS, pH 7, 80 mm KCl, 5 mm MgCl2, 0.2 mm CaCl2, 0.2 mm EGTA, variable concentrations of TG, 5 μg of microsomal protein/ml, 5 mm potassium oxalate, and 3 mm ATP. The reaction was started (37 °C) by the addition of oxalate and ATP, and was terminated at sequential times by vacuum filtration (0.45-μm Millipore filters). The filters containing the calcium loaded vesicles were washed with 2 mmLaCl3 and 10 mm MOPS, pH 7.0, and were then processed for determination of radioactivity by scintillation counting. The observed rates of Ca2+ transport were corrected to reflect the level of expressed ATPase in each microsomal preparation, as revealed by immunoreactivity and with reference to microsomes obtained from COS-1 cells transfected with wild-type SERCA-1 cDNA. ATPase activity was assayed in a reaction mixture containing 20 mm MOPS, pH 7.0, 80 mm KCl, 3 mmMgCl2, 0.2 mm CaCl2, 5 mm sodium azide, 20 μg of microsomal protein/ml, 3 μm ionophore A23187, 3 mm ATP, and 0–1000 nm TG. Ca2+-independent ATPase activity was assayed in the presence of 2 mm EGTA and no added Ca2+. The reaction was started (37 °C) by the addition of ATP, and samples were taken at serial times for determination of Pi by the method of Lanzetta et al. (21Lanzetta P.A. Alvarez L.J. Reinsch P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1821) Google Scholar). The Ca2+-dependent activity was calculated by subtracting the Ca2+-independent ATPase from the total ATPase and was corrected to account for the level of expressed protein in each microsomal preparation as revealed by immunoreactivity, and with reference to microsomes obtained from COS-1 cells transfected with wild-type SERCA-1 cDNA. The chimerization scheme for analysis of the S3 stalk segment is shown in TableI. In part A (top and bottom lines), nine amino acids of the SERCA S3 segment and of the corresponding Na+,K+-ATPase segment are aligned according to Norregaard et al. (1Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar). In the intervening lines, it is then shown how eight amino acids of the Ca2+ ATPase sequence were stepwise mutated to complement the conserved Phe256, and yield a nine-amino acid chimeric sequence identical to that of the Na+,K+-ATPase. Seven mutants were derived from this stepwise procedure and processed for transgenic expression in COS-1 cells.Table IMutation schemes for the S3 (A) and M3 (B) segmentsA.Sequence no.254255256257258259260261262Ca2+ATPaseAspGluPheGlyGluGlnLeuSerLysMutant S3,2GluHisPheMutant S3,3GluHisPheIleMutant S3,4GluHisPheIleHisMutant S3,5GluHisPheIleHisLeuMutant S3,6GluHisPheIleHisLeuIleMutant S3,7GluHisPheIleHisLeuIleThrMutant S3,8GluHisPheIleHisLeuIleThrGlyNa+,K+-ATPaseGluHisPheIleHisLeuIleThrGlyB.Sequence no.269270271272273274275276277Ca2+ATPaseValAlaValTrpLeuIleAsnIleGlyMutant M3–5SerPhePheSerIleNa+,K+-ATPaseValSerPhePheIleLeuSerIleIleThe native sequences of the S3 (stalk) and M3 (transmembrane) segments of SERCA-1 (17Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar, 25MacLennan D.H. Brandl B. PubMed Scopus Google Scholar) and of the corresponding segments of the Na+,K+-ATPase J. J.B. PubMed Scopus Google Scholar, B. FEBS Lett. 1992; PubMed Scopus Google Scholar) are aligned (top and according to Norregaard et A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar). In the S3 segment one out of nine amino acids is identical in the two and eight are to stepwise mutation from the Ca2+ to the Na+,K+-ATPase sequence to yield a nine amino acid segment of homology In the M3 segment out of nine amino acids are and five are mutated from the Ca2+ to the Na+,K+-ATPase sequence to yield a nine amino acid segment of homology in a The native sequences of the S3 (stalk) and M3 (transmembrane) segments of SERCA-1 (17Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar, 25MacLennan D.H. Brandl B. PubMed Scopus Google Scholar) and of the corresponding segments of the Na+,K+-ATPase J. J.B. PubMed Scopus Google Scholar, B. FEBS Lett. 1992; PubMed Scopus Google Scholar) are aligned (top and according to Norregaard et A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar). In the S3 segment one out of nine amino acids is identical in the two and eight are to stepwise mutation from the Ca2+ to the Na+,K+-ATPase sequence to yield a nine amino acid segment of homology In the M3 segment out of nine amino acids are and five are mutated from the Ca2+ to the Na+,K+-ATPase sequence to yield a nine amino acid segment of homology part a similar chimerization scheme for analysis of the M3 transmembrane mutations were produced to yield a nine-amino acid chimeric sequence residues of native chimerization scheme was to the S4 segment, and involved mutation of five amino acids to complement amino acids and in the native yielding a chimeric sequence that is to the corresponding segment of the Na+,K+-ATPase scheme for the S4 native sequence of the S4 (stalk) segments of SERCA-1 (17Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar, 25MacLennan D.H. Brandl B. PubMed Scopus Google Scholar) and of the Na+,K+-ATPase J. J.B. PubMed Scopus Google Scholar, B. FEBS Lett. 1992; PubMed Scopus Google Scholar) are aligned (top to by the residues amino acids are and are in the native residues were mutated from the Ca2+ to the Na+,K+-ATPase to a 25-amino acid segment in a The native sequence of the S4 (stalk) segments of SERCA-1 (17Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar, 25MacLennan D.H. Brandl B. PubMed Scopus Google Scholar) and of the Na+,K+-ATPase J. J.B. PubMed Scopus Google Scholar, B. FEBS Lett. 1992; PubMed Scopus Google Scholar) are aligned (top to by the residues amino acids are and are in the native residues were mutated from the Ca2+ to the Na+,K+-ATPase to a 25-amino acid segment In addition to the chimeric exchanges described more discrete mutations were produced in the S3 segment in and in the to the effects of in this of single mutations on and mutations on the → → → → → → mutations of the SERCA-1 to the corresponding Na+,K+-ATPase as well as single or mutations in the more as to the corresponding Na+,K+-ATPase were and effects on the sensitivity of the enzyme to TG were as shown in and The are the used to the inhibition each and to the TG 50% inhibition. in a mutations of the SERCA-1 to the corresponding Na+,K+-ATPase as well as single or mutations in the more as to the corresponding Na+,K+-ATPase were and effects on the sensitivity of the enzyme to TG were as shown in and The are the used to the inhibition each and to the TG 50% inhibition. of the COS-1 cells transfected under and the Ca2+ ATPase to the as shown following (18Zhang Z. Sumbilla C. Lewis D. Summers S. Klein M.G. Inesi G. J. Biol. Chem. 1995; 270: 16283-16290Abstract Full Text Full Text PDF PubMed Google Scholar). In the Western analysis of microsomal obtained from the cells revealed similar of expression for the wild-type ATPase and ATPase mutants of expression were to the of transfection the presence of the expression were by of Western and the were used to the functional to described with reference to the wild-type enzyme. by and K. D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar), microsomal vesicles obtained from transfected COS-1 cells ATP-dependent Ca2+ uptake and ATPase These are specific and useful functional as shown in at rates for several Ca2+ uptake is a highly specific functional is inhibited by TG 2 the other the observed ATPase activity Ca2+-independent and that from the total in order to the specific Ca2+-dependent and ATPase activity 2 the Ca2+ transport of wild-type and mutant proteins that the transport rates are by mutations of up to amino acids in the S3 segment and a as the of mutated amino acids in the S3 segment is to the other the nine-amino acid chimeric homology with the Na+,K+-ATPase in M3 and the 25-amino acid homology in S4 produce and of the transport effects of mutations were observed on the ATPase rates not We then the TG concentration sensitivity of wild-type enzyme and of mutants at 50% is shown in A that the for Ca2+ transport inhibition by TG is by 3 orders of magnitude to concentrations to as the of mutated amino acids in the M3 segment is to five or to yield a or acid segment between and Na+,K+-ATPase. were observed for Ca2+-dependent ATPase activity in the of Norregaard et al. (1Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar), the ATPase sensitivity to TG was following chimeric involving the entire S3-M3 segment, produced a nine-amino acid chimeric exchange the M3 transmembrane segment, to with the chimeric exchange produced in the S3 stalk We found that the chimeric exchange in the M3 segment produced approximately transport inhibition no effect on the ATPase sensitivity to TG the of mutations in the S3 segment with to TG then a acid chimeric mutation in the S4 segment of the ATPase in out that the segment for studies and amino with the corresponding segment of the Na+,K+-ATPase mutation of five amino acids in a acid chimeric is of that this S4 mutant inhibition of and its TG sensitivity is identical to that of the wild-type enzyme that a acid mutation in the S3 stalk segment the ATPase sensitivity to TG by 3 orders of then produced more discrete mutations to the effects of in this We found that a single mutation of to the corresponding Na+,K+-ATPase is by to the ATPase sensitivity to TG by 1 order of magnitude The of is also revealed by the of TG sensitivity observed the → mutation in the is to the more → mutation. to is produced by a involving the → mutation to the more → mutation is not obtained if is mutated to TG global inhibition and Ca2+ of whereas no inhibition is produced by TG on the Na+,K+-ATPase J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar). chimeric studies to on the TG binding that of a SERCA sequence with the corresponding Na+,K+-ATPase sequence would with inhibition. Large chimeric exchanges produce total inhibition of activity and Ca2+ transport. Nevertheless, formation of Ca2+-dependent is by chimeric proteins on it was shown that large chimeric exchanges in the SERCA region do not with inhibition by TG (15Sumbilla 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). In contrast, chimeric exchange of a acid S3-M3 segment (1Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar) the effect of TG concentrations as high as 2 μm TG and of the enzyme and chimeric shown in high high low low high of of A was obtained from Sumbilla et al. (15Sumbilla 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), from Norregaard et al. (1Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar), and and from the studies was derived from and Inesi S. Inesi G. 1997; PubMed Scopus Google Scholar). in a of A was obtained from Sumbilla et al. (15Sumbilla 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), from Norregaard et al. (1Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar), and and from the studies was derived from and Inesi S. Inesi G. 1997; PubMed Scopus Google Scholar). In produced small chimeric changes by stepwise site-directed mutations in the S3 stalk segment and the SERCA mutants with to to Ca2+ transport and and sensitivity to TG. of up to amino acids in the S3 stalk segment yield enzyme Ca2+ transport and ATPase for studies of inhibition by TG. We then found that small chimeric mutations of the S3 segment and produce a of the ATPase sensitivity to TG. mutation of → by TG the effect is obtained in that chimeric mutations in the S3 segment the ATPase affinity for TG, do not with the is of that the SERCA S4 segment in its native a high and unique of structural homology with the Na+,K+-ATPase as well as other a homology that this segment as a structural for functional of the catalytic in the and the binding in the region (18Zhang Z. Sumbilla C. Lewis D. Summers S. Klein M.G. Inesi G. J. Biol. Chem. 1995; 270: 16283-16290Abstract Full Text Full Text PDF PubMed Google Scholar, C. Sumbilla C. F. L. Inesi G. PubMed Scopus Google Scholar). the other a high of homology between the SERCA and other ATPases that S4 segment is not involved in the enzyme with TG. In found that a acid to native a 25-amino acid segment identical to that of the Na+,K+-ATPase, not at with the concentration of TG inhibition 5 and the of effect of chimeric mutations in the S4 segment the of the S3 chimeric mutations that with ATPase sensitivity to TG. in the transmembrane M3 segment are also that the S3 stalk segment is specifically involved in the ATPase sensitivity to TG and suggest that the S3 segment is involved in TG binding. the reduced sensitivity to effects of the S3 mutations is because chimeric exchanges in other regions and do not affect the ATPase sensitivity to TG (1Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar, 15Sumbilla 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). is that specific of TG with the S3 segment, in addition to stacking of the TG ring structure at the membrane S. Inesi G. 1995; PubMed Scopus (16) Google Scholar), determines the high affinity of the inhibitor for the ATPase.