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Biomedical subjects

T W Loo

Publications and source records attributed to T W Loo.

At least 55 records · Page 3Linked to original sources
PubMed1994

Prolonged association of temperature-sensitive mutants of human P-glycoprotein with calnexin during biogenesis.

Mutation of amino acids located within or immediately NH2-terminal to transmembrane segment 7 of human P-glycoprotein abolished the ability of the protein to confer resistance to cytotoxic drugs. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein, and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were processed improperly, so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. Processing of the 150-kDa P-glycoprotein to the 170-kDa mature form of the enzyme for all of the mutants, except Glu707-->Ala and Tyr710-->Ala, was dramatically increased when the cells were grown at 26 degrees C. At the lower growth temperature, the mature protein was targeted to the plasma membrane, and drug efflux activity was restored. We also analyzed the mutants for possible molecular interactions that may contribute to their intracellular retention. We found that core-glycosylated forms of the wild-type and mutant P-glycoproteins were associated with the molecular chaperone calnexin. Only wild-type enzyme, however, was able to escape association with calnexin and be targeted to the plasma membrane. Prolonged association of the mutants with calnexin may be due to misfolding of the protein as evidenced by their relative short half-life of about 3 h, compared with 50 h for the wild-type enzyme. These results suggest that calnexin contributes to a quality control mechanism to retain misfolded forms of P-glycoprotein in the endoplasmic reticulum.

3T3 Cells↗
PubMed1994

Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether glycine residues in the predicted cytoplasmic loops play essential roles in the structure and function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 20 glycine residues were changed to valine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of Gly-251, -268, -269, or -781 yielded mutant proteins which were unable to confer drug resistance in transfected cells. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were improperly processed so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. The in vivo processing of mutants Gly-269 to Val and Gly-781 to Val was temperature-sensitive. When cells expressing these mutants were grown at a lower temperature (26 degrees C), the mature 170-kDa form of P-glycoprotein was the major product. Substitution of glycine with alanine at positions 251, 268, 269, or 781 yielded mutants with structural and functional characteristics similar to wild-type enzyme. Mutation of Gly-141, 187, 288, 812, or 830 to Val, altered the drug resistance profile conferred by P-glycoproteins expressed in transfected cells. All five mutations increased relative resistance to colchicine or adriamycin, without altering relative resistance to vinblastine. These results demonstrate that glycines located in the cytoplasmic loops play important roles in structure and function of P-glycoprotein.

3T3 Cells↗
PubMed1994

Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides.

P-glycoprotein consists of two homologous halves, each composed of six potential transmembrane sequences and an ATP-binding domain. The cDNA coding for human P-glycoprotein was divided in half and subcloned into separate plasmids in order to express each half as a separate polypeptide and to characterize its contribution to function. Expression of cDNAs coding for either the NH2- or COOH-terminal half-molecules in HEK 293 cells yielded products of 88 and 64 kDa, respectively. The NH2-terminal half-molecule was glycosylated, since its size decreased from 88 to 79 kDa when expressed in the presence of tunicamycin. No change was observed in the size of the COOH-terminal half-molecule when it was expressed in the presence of tunicamycin, indicating that it was not glycosylated. The cDNAs coding for each half of P-glycoprotein were transfected into NIH-3T3 cells to test for biological activity. No drug-resistant colonies were obtained when cells were transfected with cDNA coding for each half-molecule or when cells were co-transfected with both cDNAs, although stable expression of each half-molecule was detected. The inability to confer drug resistance was likely due to a defect in targeting of the half-molecules to the cell surface. Each half-molecule was then expressed in Sf9 insect cells using a baculovirus vector to allow measurement of partial function. The half-molecules exhibited ATPase activity, but their activities were not stimulated by drug substrates. Drug-stimulatable ATPase activity was present, however, when the half-molecules were expressed together. These results suggest that coupling of ATPase activity to drug binding requires interaction between both halves of P-glycoprotein.

3T3 Cells↗
PubMed1994

Interaction of rat kidney P-glycoprotein with a urinary component and various drugs including cyclosporin A.

The interaction of rat renal P-glycoprotein with various drugs and a hydrophobic component found in rat urine was studied to gain an understanding of both its transport function in kidney and its potential role in drug secretion and drug-induced nephrotoxicity. Rat kidney brush-border membranes (BBM) were photolabeled with [3H]azidopine, a calcium-channel blocker that covalently labeles P-glycoprotein. P-glycoprotein was immunoprecipitated with a rabbit polyclonal antibody against the human MDR1 protein (multidrug resistance gene class 1). The amount of [3H]azidopine incorporated into P-glycoprotein was quantitated following gel electrophoresis and fluorography. Photolabeling inhibition assays were conducted with a panel of drugs known to interact with P-glycoprotein in multidrug-resistant cells. Verapamil or quinidine [half-maximal inhibition constant (K0.5) = 1 microM], vinblastine (K0.5 = 3 microM), and doxorubicin or daunomycin (K0.5 = 10 microM) all blocked [3H]azidopine photolabeling of renal P-glycoprotein. Of the drugs tested, the immunosuppressant drug, cyclosporin A, interacted with kidney P-glycoprotein with the highest affinity (K0.5 = 50 nM). However, the cardiac glycoside, digoxin, failed to inhibit P-glycoprotein photolabeling. A hydrophobic rat urine extract prepared by reverse-phase chromatography also blocked photolabeling of renal P-glycoprotein. Our current hypothesis is that various drugs may inhibit urinary excretion of an endogenous substrate by virtue of their ability to bind with high affinity to P-glycoprotein. A hypothesis of drug-induced nephrotoxicity based on the interaction of various compounds like cyclosporin A with P-glycoprotein is presented.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Functional expression of human renal Na+/Ca2+ exchanger in insect cells.

The cDNA coding for the human renal isoform of the Na+/Ca2+ exchanger was cloned from a human embryonic kidney cell line (HEK 293) using a polymerase chain reaction strategy. It was found by sequence analysis that the cDNA encoded for a human kidney isoform of the Na+/Ca2+ exchanger. The kidney isoform is nearly identical to the human cardiac Na+/Ca2+ exchanger except for a segment of amino acids within the predicted cytoplasmic loop region of the molecule. Within this region, the kidney isoform contains a deletion of 36 amino acids, as well as a segment of 33 amino acids that is only 33% identical to the equivalent region of the cardiac exchanger. The cDNA was then tested for function by expression in insect cells. We constructed a recombinant baculovirus containing renal Na+/Ca2+ exchanger cDNA under control of the polyhedrin promoter. High levels of expression and Na+/Ca2+ exchange activity were found in Sf9 insect cells infected with the recombinant virus. Our results indicate that human kidney expresses a functional and alternatively spliced variant of the human cardiac Na+/Ca2+ exchanger.

Amino Acid Sequence↗
PubMed1993

Deletion of NH2- and COOH-terminal sequences destroys function of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum.

Deletion mutants of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS-1 cells. The mutants were expressed at levels 7- to 15-fold lower than the wild-type and were inactive. In vitro transcription-translation-insertion experiments showed that deletion of transmembrane sequences M1 and M2, but not of M8, M9, M10 or the NH2-terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1 and M2.

Animals↗
PubMed1993

Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether phenylalanine residues in predicted transmembrane sequences play essential roles in the function of human P-glycoprotein. Mutant cDNAs, in which codons for each of the 31 phenylalanine residues were changed to alanine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of either Phe-335 to Ala in transmembrane segment 6, or Phe-978 to Ala in transmembrane segment 12, drastically altered the drug resistance profile conferred by the mutant P-glycoprotein in transfected cells. Mutant Phe-335-->Ala conferred little resistance to vinblastine or actinomycin D but retained the ability to confer resistance to colchicine and adriamycin. The mutant also showed increased binding of azidopine, which could be inhibited by lower levels of vinblastine, relative to the wild-type enzyme. By contrast, mutant Phe-978-->Ala conferred little or no resistance to colchicine or adriamycin, while its ability to confer resistance to vinblastine or actinomycin D was retained. These results suggest that Phe-335 and Phe-978 play important roles in the recognition and transport of specific substrates by P-glycoprotein. Mutation of Phe-777 to Ala affected the biosynthesis of the transporter. Mutation of the other 28 phenylalanine residues yielded protein products with structural and functional characteristics that were indistinguishable from the wild-type enzyme.

3T3 Cells↗
PubMed1993

Functional consequences of alterations to hydrophobic amino acids located in the M4 transmembrane sector of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Those hydrophobic residues between Ile298 and Ile315 in transmembrane segment M4 of the Ca(2+)-ATPase of sarcoplasmic reticulum, not previously mutated, were mutated systematically in ways that would alter their size or polarity, and functional consequences were measured. Fourteen residues in this sequence are organized as juxtapositions of large, hydrophobic (Val, Leu, Ile) and small (Ala, Gly) residues, and these were altered so that large residues were substituted for small and vice versa. Several mutants exhibited diminished Ca2+ transport, but mutants A305V and A306V lost all Ca2+ transport function. In both cases, the mutants were phosphorylated with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that the Ca(2+)-binding sites were intact. Reduced Ca2+ affinity, as measured by Ca2+ dependence of phosphorylation from ATP, was observed for mutant A305V. In both mutants, the ADP-insensitive phosphoenzyme intermediate (E2P) decayed slowly relative to the wild-type enzyme, suggesting that the E2P to E2 conformational transition was impaired, slowing the rate of the phosphatase reaction. Double mutants which reversed the order of Val304 and Ala305 and Ala306 and Ile307, resulted in the same phenotype as the single Ala mutations. These results, combined with our previous demonstration that Glu309 is a Ca2+ binding residue, that Pro312 is involved in E1P to E2P conformational changes, and that Gly310 is involved in E2P to E2 conformational changes, support the hypothesis that transmembrane segment M4 plays a key role in the Ca2+ transport function of the Ca(2+)-ATPase through its involvement in both the binding of Ca2+ and the subsequent conformational changes which bring about the translocation of Ca2+ to the lumen of the membrane.

Adenosine Triphosphate↗
PubMed1993

Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether prolines in the predicted transmembrane domains play essential roles in the function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 13 prolines were changed to alanine were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutations of either Pro223 in transmembrane segment 4 or Pro866 in transmembrane segment 10, drastically reduced the ability of the mutant proteins to confer resistance to colchicine, adriamycin, or actinomycin D, whereas the capacity to confer resistance to vinblastine was retained. These results strongly suggest that residues in putative transmembrane segments 4 and 10, which are found in identical positions when homologous, presumably duplicated, halves of the transporter are aligned, play important roles in recognition of colchicine, adriamycin, and actinomycin D. They may either interact to form a single drug-binding site or form part of two equivalent, but independent, drug-binding sites. The lack of detectable effect of either mutation on vinblastine transport, however, indicates that there are differences in the requirements for binding of various substrates to P-glycoprotein. Mutation of Pro709 in transmembrane segment 7 resulted in a protein unable to confer drug resistance. A change at this position was found to induce a structural aberration, since the major protein product observed in transfected cells had an apparent molecular weight of 150,000, whereas the wild-type enzyme had an apparent molecular weight of approximately 170,000. Mutation of the other 10 prolines yielded protein products with structural and functional characteristics indistinguishable from wild-type P-glycoprotein.

3T3 Cells↗
PubMed1992

Site-directed mutagenesis of the Ca2+ ATPase of sarcoplasmic reticulum.

We have cloned cDNA encoding the Ca2+ ATPase from fast-twitch skeletal muscle and, on the basis of its amino acid sequence and immunological studies of its topology, have made deductions concerning its secondary structure and active sites. These deductions have led us to test models for Ca2+ transport through expression of the protein in functional form in COS-1 cells, mutagenesis, and measurement of altered function. Mutation of about 250 of the 1000 amino acids making up the Ca2+ pump has indicated that the sites of high affinity Ca2+ binding are located in the center of the transmembrane domain and are made up from residues located in transmembrane sequences M4, M5, M6 and M8. The ATP binding site appears to be located in the headpiece and is made up from a series of loop sequences connecting alternating alpha helices and beta strands. Sites of conformational interaction appear in all domains throughout the Ca2+ pump. In our present model, Ca2+ transport occurs through binding to high affinity sites accessible to the cytoplasm in the E1 conformation, followed by release to the lumen from low affinity sites which form during the ATP-induced transition of the protein from the E1 to the E2 conformation.

Animals↗
PubMed1990

Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.

Amino acids in three highly conserved segments of the Ca2(+)-ATPase. Asp-Pro-Pro-Arg604, Thr-Gly-Asp627, Thr-Gly-Asp703 as well as Asp707, have been proposed to participate in formation of the nucleotide binding site. We have tested this hypothesis by investigating the properties of mutants with alterations to amino acids within these segments. Most of the mutants were found to be defective in Ca2+ transport function. The inactive mutants could be separated into two classes on the basis of the kinetics of phosphoenzyme intermediate formation and decomposition. One group, Asp601----Asn, Pro603----Leu, Asp627----Glu, and Asp703----Asn, formed phosphoenzyme intermediates with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that both the high affinity Ca2+ binding sites and the nucleotide binding sites were intact. In each of these mutants, however, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive phosphoenzyme intermediate very slowly, relative to the wild-type enzyme. Thus the inability of these mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. Another group, Asp601----Glu, Pro603----Gly, Asp707----Glu, and Asp707----Asn, did not form detectable phosphoenzyme intermediates from either ATP or Pi. Although we have demonstrated an effect on Ca2+ transport of mutations in each of the highly conserved regions predicted to be involved in ATP binding, we cannot yet define their roles in ATP-dependent Ca2+ transport.

Adenosine Triphosphate↗
PubMed1990

The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the luminal surface of the Ca2(+)-ATPase of sarcoplasmic reticulum.

The epitope for monoclonal antibody A20 was mapped to amino acids 870-890 of the Ca2(+)-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The antibody did not react with the epitope in intact sarcoplasmic reticulum vesicles but reacted with the epitope when the vesicles were solubilized with the detergent C12E8 or made permeable by incubation in a hypotonic medium. By contrast, antibody A52, which binds to a cytoplasmic epitope consisting of amino acids 657-672, reacted with the Ca2(+)-ATPase in vesicular, permeabilized vesicular, and C12E8-solubilized states. These results clearly demonstrate that antibody A20 binds to a luminal epitope and provide the first demonstration that a specific segment of the Ca2(+)-ATPase is located on the luminal surface of the sarcoplasmic reticulum. These results are consistent with, and support, our model for folding of the Ca2(+)-ATPase (Brandl, C. J., Korczak, B., Green, N. M., and MacLennan, D. H. (1986) Cell 44, 597-607) in which residues 657-672 were proposed to form part of the cytoplasmic nucleotide binding domain, while residues 870-890 were proposed to form a luminal loop between proposed transmembrane sequences M7 and M8.

Animals↗
PubMed1990

Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.

The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.

Amino Acid Sequence↗
PubMed1990

Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.

Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 (ligands 1 to 6, respectively) appear to form the high affinity Ca2(+)-binding sites of the Ca2(+)-ATPase. The plasticity of the Ca2(+)-binding sites was tested by separate replacement of each of the ligands with a structurally similar oxygen-containing residue using site-specific mutagenesis. Mutant cDNAs were transfected into COS-1 cells, and ATP-dependent Ca2+ transport or partial reactions were studied in microsomes containing the expressed Ca2(+)-ATPases. In most cases where amino acid substitutions were carried out, the expressed enzymes lacked Ca2+ transport function and Ca2(+)-dependent phosphorylation by ATP. Furthermore, the mutant enzymes were phosphorylated by inorganic phosphate, even in the presence of Ca2+, which inhibits phosphorylation of the wild-type enzyme possessing intact Ca2(+)-binding sites. On mutant, however, containing an isosteric replacement of Glu by Gln at ligand 6, exhibited wild-type levels of Ca2+ transport activity and Ca2+ affinity. Two mutants exhibited properties consistent with a reduction in Ca2+ affinity. In the mutant in which Thr was replaced by Ser at ligand 4, Ca2+ transport activity was 70% of wild-type, while half-maximal activation by Ca2+ occurred at 0.8 microM as compared to 0.3 microM for the wild-type enzyme. In the mutant Glu309----Asp at ligand 1, Ca2+ transport activity was lost, but Ca2(+)-activated phosphorylation by ATP was retained. The concentration of Ca2+ required to activate phosphorylation was increased about 10-fold, however, compared to wild type. These results support our hypothesis that ligands 1 to 6, believed to reside within the transmembrane domain, interact with Ca2+ ions during the transport process. The roles of 12 other oxygen-containing residues and of His278 located in the transmembrane domain were also examined by mutation. Although the oxygen-containing side chains of these residues are potential Ca2+ ligands, their replacement with nonpolar amino acids did not abolish Ca2+ transport function, leading to the conclusion that they are not essential ligands for high affinity Ca2+ binding by the Ca2+ pump.

Amino Acid Sequence↗
PubMed1989

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum.

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.

Amino Acid Sequence↗
PubMed1989

Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum.

Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+ transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+ transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+ transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+ transport activity to alterations of Asn111 and Asn114. The mutant Asn114 to alanine retained about 50% activity, whereas the change Asn111 to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+ uptake observed in the Asn111 and Asn114 mutants was not due to a defect in enzyme activation by Ca2+ or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+ following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+ and to channel it to domains that are involved in enzyme activation and cation translocation. Simultaneous mutation of 4 glutamate residues to alanine in the lumenal loop between transmembrane sequences M1 and M2 did not affect Ca2+ transport activity, indicating that acidic residues in this lumenal loop do not play an essential role in Ca2+ transport. Similarly, mutation of Glu192 and Asp196 in the beta-strand domain between stalk helices 2 and 3 did not affect Ca2+ transport activity, although mutation of Asp196 did diminish expression of the protein.

Amino Acid Sequence↗
PubMed1989

Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase.

Cation pumps bind and translocate ions with the intermediate formation of a phosphoenzyme. In spite of extensive knowledge of the primary and even secondary structures of several of these cation transport enzymes, however, no high affinity cation binding sites have yet been determined. Here we report the use of oligonucleotide-directed, site-specific mutagenesis to identify the amino acids involved in Ca2+ binding in one of these transport enzymes, the Ca2+-ATPase of sarcoplasmic reticulum. Alteration of Glu 309, Glu 771, Asn 796, Thr 799, Asp 800 or Glu 908, each of which is predicted to lie near the centre of the transmembrane domain in putative transmembrane sequences M4, M5, M6 and M8 resulted in complete loss of Ca2+ transport function and of Ca2+-dependent phosphorylation of the enzyme by ATP. Phosphorylation of each of the mutant enzymes with inorganic phosphate was observed, however, even in the presence of Ca2+, which inhibits phosphorylation in the wild-type enzyme possessing an intact high affinity Ca2+-binding site. These results suggest that at least six polar, oxygen-containing residues lying near the centre of the transmembrane domain provide ligands for one or both of the two high affinity Ca2+ binding sites in the Ca2+-ATPase.

Animals↗