Structure-function relationships in sarcoplasmic or endoplasmic reticulum type Ca2+ pumps.
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Animals↗
Biomedical subjects
D H MacLennan
Publications and source records attributed to D H MacLennan.
Characterization of a Ca2+ binding and regulatory site in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.
A region in the skeletal muscle ryanodine receptor between amino acids 4014 and 4765 was expressed as a trpE fusion protein. Overlay studies revealed that this region bound Ca2+ and ruthenium red, an indicator of Ca(2+)-binding sites. Ca2+ binding was mapped to subregion 13b between amino acids 4246 and 4377, encompassing a predicted high affinity Ca(2+)-binding site, and to subregion 13c between amino acids 4364 and 4529, encompassing two predicted high affinity Ca(2+)-binding sites. Ca2+ binding was then mapped to three shorter sequences, 22(13b1), 36(13c1), and 35(13c2), amino acids long, each encompassing one of the three predicted Ca(2+)-binding sites. Site-directed polyclonal antibodies were raised against these three short sequences and purified on antigen affinity columns. The antibody against sequence 13c2, lying between residues 4478 and 4512, specifically recognized both denatured and native forms of the ryanodine receptor, suggesting that at least part of the 35 amino acid sequence containing the Ca(2+)-binding site is surface-exposed. The affinity purified antibody increased the Ca2+ sensitivity of ryanodine receptor channels incorporated into planar lipid bilayers, resulting in increased open probability and opening time without altering channel conductance. The antibody-activated channel was still modulated by Ca2+, Mg2+, ATP, ryanodine, and ruthenium red. These observations suggest that sequence 13c2 may be involved in Ca(2+)-induced Ca2+ release.
Animals↗
Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.
ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.
Adenosine Triphosphate↗
The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases.
Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.
Animals↗
Malignant hyperthermia.
In humans genetically predisposed to malignant hyperthermia, anesthesia can induce skeletal muscle rigidity, hypermetabolism, and high fever, which, if not immediately reversed, can lead to tissue damage or death. The corresponding condition in swine leads to stress-induced deaths and devalued meat products. Abnormalities in the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been implicated in the cause of both the porcine and human syndromes by physiological and biochemical studies and genetic linkage analysis. In swine, a single founder mutation in the ryanodine receptor gene (RYR1) can account for all cases of malignant hyperthermia in all breeds, but a series of different RYR1 mutations are likely to be uncovered in human families with MH. Moreover, lack of linkage between malignant hyperthermia and RYR1 in some families indicates a heterogeneous genetic basis for the human syndrome.
Animals↗
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↗
The genetic basis of malignant hyperthermia.
Anaesthesia can induce skeletal muscle rigidity, hypermetabolism and high fever in humans genetically predisposed to malignant hyperthermia. If not immediately reversed, such episodes can lead to tissue damage and death. In swine with the corresponding condition, stress can induce death or lead to devalued meat products. Since muscle contraction is controlled by sarcoplasmic Ca2+, the abnormality, as reviewed here by David H. MacLennan, could reside in the skeletal muscle Ca(2+)-release channel gene, RYR1. Several observations support the view that a single RYR1 mutation is causal of malignant hyperthermia in all breeds of pigs and in at least some human families: the substitution of Cys for Arg615 as the sole deduced amino acid sequence change in a comparison of malignant hyperthermia and normal porcine RYR1 cDNAs; the linkage of this mutation to malignant hyperthermia in over 450 pigs in six breeds, including 338 meioses; and the appearance of the corresponding mutation, Cys for Arg614, across a species barrier, in a few human families, where it also cosegregates with malignant hyperthermia. Linkage of malignant hyperthermia to RYR1 is, however, not observed in all human families with malignant hyperthermia. Accordingly, other abnormal genes that may cause the condition are being sought.
Anesthesia↗
Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.
Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.
Amino Acids↗
Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia.
The substitutions of T for C1843 in the porcine ryanodine receptor (RYR1) gene, which deletes a HinPI restriction endonuclease site and creates a HgiAI site, and of T for C1840 in human RYR1, which deletes a RsaI site, lead to Cys for Arg substitutions in the ryanodine receptors and are probable causal mutations for malignant hyperthermia (MH). To improve the restriction endonuclease assay of these sites, thereby providing an accurate, reliable diagnosis for MH, introns flanking the exon containing the mutation were sequenced, permitting identification and PCR amplification of a 659-bp porcine gene sequence that contains both constant and variant HgiAI sites and a 922-bp human gene sequence that contains both constant and variant RsaI sites. As a result, these PCR-amplified sequences contain constant internal controls for the reliable differentiation by restriction endonuclease digestion of normal, heterozygous, and MH genotypes.
Animals↗
Structure-function relationships in the Ca2+ pump of the sarcoplasmic reticulum.
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Adenosine Triphosphate↗
Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development.
The expression of major sarcoplasmic reticulum proteins during cardiac and fast-twitch skeletal muscle development was examined using gene-specific probes. Through the use of S1 nuclease mapping, Northern blot, and RNA slot-blot analysis, sarcoplasmic reticulum proteins were shown to exhibit both narrow tissue specificity and plasticity in their expression during muscle development. In fast-twitch skeletal muscle, the cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin were detected at high levels in fetal stages but were gradually replaced by fast-twitch isoforms in adult muscle. In contrast, cardiac muscle expressed exclusively cardiac/slow-twitch isoforms of Ca(2+)-ATPase and calsequestrin at all stages. Both fast-twitch and slow-twitch skeletal muscle expressed the same skeletal muscle ryanodine receptor isoform, whereas cardiac muscle expressed a cardiac isoform. Phospholamban expression was restricted to cardiac and slow-twitch skeletal muscle and did not appear in developing fast-twitch skeletal muscle. During in vitro myogenesis of C2C12 cells, the mRNA transcripts encoding sarcoplasmic reticulum proteins were found to be coordinately induced in synchrony with that of contractile protein mRNA. The myogenic factor "myogenin" induced sarcoplasmic reticulum gene transcripts along with contractile protein mRNAs in nonmyogenic cells. These data suggest that the induction of both sarcoplasmic reticulum and contractile protein gene families is under the control of a common myogenic differentiation program.
Actins↗
The role of the skeletal muscle ryanodine receptor gene in malignant hyperthermia.
Malignant hyperthermia (MH) is an inherited, potentially lethal condition in which sustained muscle contracture with attendant hypermetabolism and hyperthermia is triggered in humans, heterozygous for the gene defect, by inhalational anaesthetics and skeletal muscle relaxants, and in pigs, homozygous for the defect, by stress. Because muscle contracture could result from a defective Ca2+ release channel, we have focussed our attention on the linkage of MH to defects in the gene (RYR1) encoding the skeletal muscle Ca2+ release channel. We have cloned and sequenced human RYR1 cDNA and found restriction fragment length polymorphisms (RFLPs) in the human gene. We also localized RYR1 to human chromosome 19q13.1. Studies of the cosegregation of MH with these RFLPs established RYR1/MH linkage on human chromosome 19q13.1 (lod score of 4.2; recombinant fraction 0.0). We then sequenced MH and normal porcine RYR1 cDNAs. Mutation of C1843 to T, leading to substitution of Cys for Arg615, was the sole amino acid change noted between MH and normal animals. Linkage of this mutation to MH was established in a study of 338 informative meioses (lod score of 102; recombinant fraction 0.0). We identified the corresponding mutation in 1 of 35 human MH families studied and found cosegregation of the mutation and MH. The combination of a high lod score with crossing of a species barrier supports the causal nature of this mutation. Future studies are aimed at finding the major human MH mutations and establishing assays for their accurate diagnosis.
Amino Acid Sequence↗
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↗
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↗
Evidence for genetic heterogeneity of malignant hyperthermia susceptibility.
A locus for malignant hyperthermia susceptibility (MHS) has been localized on chromosome 19q12-13.2, while at the same time the gene encoding the skeletal muscle ryanodine receptor (RYR1) also has been mapped to this region and has been found to be tightly linked to MHS. RYR1 was consequently postulated as the candidate for the molecular defect causing MHS, and a point mutation in the gene has now been identified and is thought to be the cause of MH in at least some MHS patients. Here we report the results of a linkage study done with 19q12-13.2 markers, including the RYR1 cDNA, in two Bavarian families with MHS. In one of the families, three unambiguous recombination events between MHS and the RYR1 locus were found. In the second family only one informative meiosis was seen with RYR1. However, segregation analysis with markers for D19S75, D19S28, D19S47, CYP2A, BCL3, and APOC2 shows that the crossovers in the first family involve the entire haplotype defined by these markers flanking RYR1 and, furthermore, reveals multiple crossovers between these haplotypes and MHS in the second family. In these families, pairwise and multipoint lod scores below -2 exclude MHS from an interval spanning more than 26 cM and comprising the RYR1 and the previously described MHS locus. Our findings thus strongly suggest genetic heterogeneity of the MHS trait and prompt the search for another MHS locus.
Adult↗
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↗
Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.
Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate 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 after 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. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as 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. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphate↗
Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia.
Malignant hyperthermia (MH) causes neurological, liver, and kidney damage and death in humans and major economic losses in the swine industry. A single point mutation in the porcine gene for the skeletal muscle ryanodine receptor (ryr1) was found to be correlated with MH in five major breeds of lean, heavily muscled swine. Haplotyping suggests that the mutation in all five breeds has a common origin. Assuming that this is the causal mutation for MH, the development of a noninvasive diagnostic test will provide the basis for elimination of the MH gene or its controlled inclusion in swine breeding programs.
Amino Acid Sequence↗