Biomedical subjects

B Vilsen

Publications and source records attributed to B Vilsen.

At least 37 records · Page 2Linked to original sources

PubMed1994

Alteration of intracellular Ca2+ transients in COS-7 cells transfected with the cDNA encoding skeletal-muscle ryanodine receptor carrying a mutation associated with malignant hyperthermia.

Malignant hyperthermia (MH), an inherited neuromuscular disease triggered by halogenated inhalational anaesthetics and skeletal-muscle relaxants, appears to be due to an alteration of intracellular Ca2+ homoeostasis. MH occurs in 1 out of 20,000 anaesthetized adults and is characterized by hypermetabolism, skeletal-muscle rigidity and elevation in body temperature, which is frequently fatal [MacLennan and Phillips (1992) Science 256, 789-794]. The defect responsible for the disease may lie within the mechanism controlling the release of Ca2+ from sarcoplasmic reticulum via the ryanodine-receptor (RYR) Ca2+ channel; in fact a point mutation in the RYR has been associated with MH in some human families, as well as in the MH-susceptible pig. To date, however, no direct evidence has been obtained demonstrating that the point mutation is both necessary and sufficient to cause functional alterations in RYR-mediated Ca2+ release. In the present report we show that the presence of the Arg-to-Cys point mutation in the recombinant RYR expressed in COS-7 transfected cells causes abnormal cytosolic Ca2+ transients in response to 4-chloro-m-cresol, an agent capable of eliciting in vitro contracture of MH-susceptible muscles.

Amino Acid Sequence↗

PubMed1994

Amino acids Asn796 and Thr799 of the Ca(2+)-ATPase of sarcoplasmic reticulum bind Ca2+ at different sites.

The Ca(2+)-binding properties of mutants Asn796-->Ala, Thr799-->Ala, and Glu908-->Ala of the sarcoplasmic reticulum Ca(2+)-ATPase were analyzed in studies of CrATP-induced Ca2+ occlusion and of the Ca2+ dependencies of phosphorylation from ATP and P(i). The Asn796-->Ala and Thr799-->Ala mutants were unable to occlude Ca2+, whereas the Glu908-->Ala mutant was fully capable of occluding Ca2+. Mutant Asn796-->Ala was unable to form a phosphoenzyme from ATP, whereas mutants Thr799-->Ala and Glu908-->Ala phosphorylated from ATP with K0.5 values for Ca2+ activation of 3 and 0.4 mM, respectively. In the Asn796-->Ala mutant, Ca2+ inhibited phosphorylation from P(i) with a K0.5 value of less than 10 microM, whereas in mutants Thr799-->Ala and Glu908-->Ala, the K0.5 values for Ca2+ inhibition of phosphorylation from P(i) were much higher and similar to the K0.5 values observed for Ca2+ activation of phosphorylation from ATP. These data are consistent with a model in which the respective side chains of Asn796 and Thr799 are assigned to two different Ca2+ sites being involved in Ca2+ occlusion, while the side chain of Glu908 is excluded as a direct Ca2+ ligand in the Ca2+ occluded complex. The dephosphorylation of the ADP-insensitive E2P phosphoenzyme intermediate was blocked in the Asn796-->Ala mutant, suggesting the possibility that Asn796 participates in the countertransport of protons.

Amino Acid Sequence↗

PubMed1993

Chimeric Ca(2+)-ATPase/Na+,K(+)-ATPase molecules. Their phosphoenzyme intermediates and sensitivity to Ca2+ and thapsigargin.

Chimeric molecules consisting of parts from the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase were expressed in COS-1 cells and analysed functionally. One chimera, in which most of the central cytoplasmic loop was derived from the Na+,K(+)-ATPase, while the transmembrane segments and the minor cytoplasmic loop came from the Ca(2+)-ATPase, was able to occlude Ca2+ and to be phosphorylated from ATP with normal apparent affinity for Ca2+ and ATP. This chimera also displayed normal sensitivity to thapsigargin, but was unable to undergo the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and to transport Ca2+. The other chimera, which consisted of the NH2-terminal two-thirds of Na+,K(+)-ATPase and the COOH-terminal one-third of Ca(2+)-ATPase, was unable to phosphorylate from ATP, but phosphorylated from inorganic phosphate in a Ca(2+)-inhibitable and thapsigargin-insensitive reaction. These results can be explained in terms of a structural model in which the non-conserved residues in the central cytoplasmic domain of the Ca(2+)-ATPase are without major importance for the binding and occlusion of Ca2+, but are involved in the E1P-->E2P conformational changes of the phosphoenzyme, whereas residues in transmembrane segments on both sides of the central cytoplasmic domain are involved in formation of the Ca(2+)-binding sites. The data moreover show that thapsigargin sensitivity is dependent on residues in the NH2-terminal one-third of the Ca(2+)-ATPase molecule.

Adenosine Triphosphate↗

PubMed1993

Glutamate 329 located in the fourth transmembrane segment of the alpha-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions.

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G to C base substitution in the cDNA, which on the amino acid level gave rise to a glutamine in place of the glutamate residue Glu329, previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. Functional analysis on isolated plasma membranes demonstrated that the Glu329-->Gln variant was able to catalyze Na(+)- and K(+)-activated ATPase activity with a maximum turnover number similar if not identical to that of the wild type, but the variant exhibited a reduced affinity for both cations corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold increase in K0.5 for K+. Moreover, the apparent affinity for ATP was increased 15-fold in the variant relative to wild-type Na+,K(+)-ATPase. The Na+,K(+)-ATPase activity of the variant displayed an anomalous pH dependence with a down-shift of the pH optimum and a nearly constant rate in the range between pH 7.0 and 8.7.

Adenosine Triphosphate↗

PubMed1993

A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number.

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.

Adenosine Triphosphate↗

PubMed1993

Functional consequences of substitution of the seven-residue segment LysIleArgAspGlnMetAla240 located in the stalk helix S3 of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-directed mutagenesis was used to substitute the seven-residue segment LysIleArgAspGlnMetAla240 located at the NH2- terminal end of the "stalk" helix S3, near the beta-strand domain, in the sarcoplasmic reticulum Ca(2+)-ATPase of rabbit fast twitch muscle, with the corresponding Na+,K(+)-ATPase segment ArgIleAlaThrLeuAlaSer. This led to a new phenotypic variant of Ca(2+)-ATPase. The overall turnover rates for Ca2+ transport and ATP hydrolysis measured at 27 and 37 degrees C, respectively, were reduced to 30-40% of the wild-type rates. Analysis of the phosphoenzyme intermediates at 0 degrees C showed that the ADP-insensitive phosphoenzyme intermediate accumulated under conditions where the ADP-sensitive phosphoenzyme intermediate predominated in the wild-type Ca(2+)-ATPase. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate formed through the forward reaction with ATP, or in the "backdoor" reaction with Pi, was reduced severalfold in the mutant relative to the dephosphorylation rate measured in the wild type, but there was no significant difference between the mutant and the wild type with respect to the apparent affinity for Pi measured under equilibrium conditions. The mutant was much less susceptible to inhibition by vanadate than the wild type, under equilibrium conditions as well as during turnover with ATP and Ca2+. These observations suggest that the transition state in the hydrolysis of the aspartyl phosphate bond in the ADP-insensitive phosphoenzyme intermediate was destabilized in the mutant.

Amino Acid Sequence↗

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↗

PubMed1992

CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.

Adenosine Diphosphate↗

PubMed1992

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.

Animals↗

PubMed1992

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

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

Adenosine Diphosphate↗

PubMed1992

Deduced amino acid sequence and E1-E2 equilibrium of the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Comparison with the Ca(2+)-ATPase of rabbit fast twitch muscle.

The cDNA encoding a Ca(2+)-transport ATPase of frog (Rana esculenta) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca(2+)-ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca(2+)-ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca(2+)-ATPase were compared with those of the rabbit fast twitch muscle Ca(2+)-ATPase by functional expression of the cDNAs in COS-1 cells. The amphibian Ca(2+)-ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated in the frog Ca(2+)-ATPase than in the mammalian enzyme.

Amino Acid Sequence↗

PubMed1992

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

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

Adenosine Triphosphate↗

PubMed1992

Molecular cloning, functional expression and tissue distribution of the cDNA encoding frog skeletal muscle calsequestrin.

We have cloned, sequenced and expressed the cDNA encoding frog skeletal muscle calsequestrin. The processed frog calsequestrin is 398 residues long, with an Mr of 45941 (unglycosylated form), and exhibits 77% sequence similarity with its rabbit counterpart. Consensus sequences for glycosylation and phosphorylation of the protein were conserved. Compared with rabbit calsequestrin, the mature amphibian protein has peculiar structural properties, which include (i) a higher content of negatively charged residues (142 versus 109), and (ii) a striking repeat sequence at the C-terminal region of 44 aspartic acid residues. Furthermore, this is the first report on the expression of calsequestrin cDNA in COS-1 cells; the expressed protein exhibited an Mr and antigenic properties which were indistinguishable from those of the native protein. In addition, it was capable of binding 45Ca in a ligand overlay. Northern blot analysis of frog skeletal muscle, liver, heart and brain RNA showed that the protein is mainly expressed in skeletal muscle. The high density of negative charges at the C-terminus might constitute high-capacity low-affinity Ca(2+)-binding sites, which may account for the higher Ca(2+)-binding capacity of frog calsequestrin compared with other members of the calsequestrin family (56 mol/mol versus 40-44 mol/mol of protein).

Amino Acid Sequence↗

PubMed1992

Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.

The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in the CrATP-enzyme complex were consistent with the existence of two nonequivalent and interdependent Ca2+ occlusion sites, both in the membranous Ca(2+)-ATPase and in a detergent-solubilized monomeric Ca(2+)-ATPase preparation. The rate constant for release of the first calcium ion was k1 = 0.99 h-1, whereas the second calcium ion was released with a rate constant of k2 = 0.25 h-1 when the first site was empty and with a rate constant of k3 = 0.13 h-1 when the first site was occupied by Ca2+. Ca2+ binding at the first site occurred with a rate constant of k-1 = 0.96 microM-1 h-1 (apparent Kd = 1.0 microM). The Ca(2+)-occluded state was further stabilized by ADP, binding in exchange with ATP with an apparent Kd of 8.6 microM. Two kinetic classes of CrATP-binding sites were observed, each with a stoichiometry of 3-4 nmol/mg of protein; but only the fast phase of CrATP binding was associated with Ca2+ occlusion. Derivatization of the Ca(2+)-ATPase with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodimide resulted in inactivation of phosphorylation of the enzyme from MgATP, whereas the ability to occlude Ca2+ in the presence of CrATP was retained, albeit with a reduced apparent affinity for Ca2+.

Adenosine Diphosphate↗

PubMed1992

Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.

Binding, Competitive↗

PubMed1992

Structural basis for the E1/E1P-E2/E2P conformation changes in the sarcoplasmic reticulum Ca(2+)-ATPase studied by site-specific mutagenesis.

Site-specific mutagenesis and functional expression of cloned cDNA of the sarcoplasmic reticulum Ca(2+)-ATPase have been used to demonstrate that exchange of single amino acid residues in the Ca(2+)-ATPase can lead to forms with either "E1/E1P" or "E2/E2P" characteristics. The localization of the mutated residues identifies the M4S4 segment of the ATPase molecule as central to the energy-tranducing conformation change, and on the basis of this information a model for the pump mechanism is discussed.

Animals↗

PubMed1992

Mutational analysis of the role of Lys684 in the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional role of Lys684 located in the "hinge-domain", a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to Arg, Ala, His and Gln resulted in complete loss of calcium transport function and ATPase activity. For the Lys684- > Ala, His, Gln mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi, whereas the Lys684- > Arg mutant retained the ability to phosphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions following phosphorylation from ATP in the Lys684- > Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. In the Lys684- > Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. These data point to a role of Lys684 in the binding and transfer of phosphate to the protein, and in the transport-associated conformational changes of the phosphorylation site.

Adenosine Triphosphate↗

PubMed1991

Functional consequences of alterations to hydrophobic amino acids located at the M4S4 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to investigate the functional roles of amino acids in the relatively hydrophobic sequence Ile-Thr-Thr-Cys-Leu-Ala-320, located at the M4S4 boundary of the sarcomplasmic reticulum Ca(2+)-ATPase. Each of the residues was replaced with either a less hydrophogic, a polar, or a charged residue. Mutants Ile-315----Arg and Leu-319----Arg were devoid of any Ca2+ transport function or ATPase activity, while the mutant Thr-317----Asp retained about 5 and 7% of the wild-type Ca2+ transport and ATPase activities, respectively. These three mutants were able to form the ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP, but this intermediate decayed very slowly to the ADP-insensitive phosphoenzyme intermediate (E2P). In the mutants Ile-315----Arg and Leu-319----Arg, the level of E2P formed in the backward reaction with inorganic phosphate was extremely low, but hydrolysis of E2P occurred at a normal rate. These mutants, in addition, displayed a higher apparent affinity for Ca2+ than the wild-type enzyme. In the mutants Ile-315----Ser and Ile-315----Asp, the Ca2+ transport and ATPase activities were moderately reduced to 30-40% of the wild-type activities, but normal affinities for Ca2+, Pi, and ATP were retained, as was the low affinity modulatory effect of ATP. Mutation of Thr-316 to Asp, Thr-317 to Ala, Cys-318 to Ala and Ala-320 to Arg had little or no effect on Ca2+ transport or ATPase activities. Introduction of two negative and one positive charge by triple mutation of the Ile-Thr-Thr-317 sequence created a mutant enzyme that, although completely inactive, was inserted into the membrane, consistent with a location of these residues on the cytoplasmic side of the M4S4 interface. Our findings suggest that the amphipathic character of the S4 helix and/or the distribution of charges in S4 is important for the stability of the E2P intermediate.

Amino Acid Sequence↗