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D H MacLennan

Publications and source records attributed to D H MacLennan.

At least 37 records · Page 2Linked to original sources
PubMed1999

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease.

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Amino Acid Sequence↗
PubMed1999

Calreticulin is essential for cardiac development.

Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.

Animals↗
PubMed1999

Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels.

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.

Animals↗
PubMed1998

Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay.

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.

Adenosine Triphosphate↗
PubMed1998

Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.

The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis. Acidic amino acids Asp3987, Glu4032, Asp4815, Asp4917, Asp4938, and Asp4969 and amidated residues Asn4034, Asn4037, Asn4574, Asn4805, Asn4806, and Gln4933, and Gly4033 were mutated to Ala, and Ala3988 was mutated to Val. When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m-cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [3H]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [3H]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1. However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [3H]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu4032 (M2), Asn4806 (M7), Asp4815 (M7), Asp4917 (M10), and Gln4933 (M10) are involved in channel function and regulation.

Amino Acid Sequence↗
PubMed1998

The cytoplasmic loops between domains II and III and domains III and IV in the skeletal muscle dihydropyridine receptor bind to a contiguous site in the skeletal muscle ryanodine receptor.

Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR). Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization-induced inactivation of RyR1. The DHPR III-IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for 35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR III-IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys954-Asp1112 retained full binding activity, whereas Leu922-Phe1075 had no binding activity. The RyR1 sequence Arg1076-Asp1112, previously shown to interact with the DHPR II-III loop (Leong, P., and MacLennan, D., H. (1998) J. Biol. Chem. 273, 7791-7794), bound to DHPR III-IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys954-Asp1112. These data suggest that the site of DHPR III-IV loop interaction contains elements from both the Lys954-Phe1075 and Arg1076-Asp1112 fragments. The presence of 4 +/- 0.4 microM GST-DHPR II-III or 5 +/- 0.1 microM His-peptide-DHPR III-IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922-Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922-Asp1112 binding to GST-DHPR II-III and GST-DHPR III-IV by His10-DHPR II-III and His-peptide-DHPR III-IV was observed. These studies indicate that the DHPR II-III and III-IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys 954 and Asp1112.

Amino Acid Sequence↗
PubMed1998

Sites of regulatory interaction between calcium ATPases and phospholamban.

Phospholamban (PLN) is a 52-amino acid, integral membrane protein that interacts with and reversibly inhibits the activity of the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a). We have used site-directed mutagenesis to analyze the sites of interaction between PLN and SERCA2a. First, we used chimera formation between SERCA2a and SERCA3 (which is weakly inhibited by PLN) to determine the interacting residues in cytoplasmic sequences of SERCA2 and PLN. Then, we expressed SERCA2a with the transmembrane sequence of PLN and demonstrated that the sites of inhibitory interaction are located in transmembrane sequences of the two proteins. We proposed that a four-base circuit involving noninhibitory cytoplasmic and inhibitory transmembrane sites in PLN and SERCA2a best describes the interaction. Recently, we have used alanine-scanning mutagenesis to show an asymmetric distribution of function in the transmembrane domain of PLN--one helical face interacts with PLN molecules in a pentamer, and the other interacts with SERCA2a. Gain of function by mutation of PLN-interacting residues indicates that the inhibitory species of PLN is a monomer. Thus regulatory steps include PLN dissociation, PLN/SERCA2a inhibitory association, and PLN/SERCA2a dissociation induced by phosphorylation of PLN (in the noninhibitory cytoplasmic domain) or by binding of Ca2+ by SERCA2a (in the inhibitory transmembrane domain).

Amino Acid Sequence↗
PubMed1998

Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum.

Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib in phospholamban (PLN), revealed that mutation to Ala of Asn27, Gln29, and Asn30 results in gain of inhibitory function. In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M. , and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), gain of function in PLN transmembrane domain II mutants was correlated with pentamer destabilization, leading to proposals that the PLN monomer is the active inhibitory species, that dissociation of the PLN pentamer is one determinant of PLN inhibitory function and that dissociation of the PLN.cardiac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a second determinant. Because each of the new domain Ib mutants contained a normal ratio of pentamer to monomer in SDS-polyacrylamide gel electrophoresis, gain of function must have resulted from mechanisms other than destabilization of pentameric structure. Evidence that domain Ib and domain II mutants act through different sites and different mechanisms was provided by a monomeric double mutant, N30A/I40A, in which the enhanced inhibitory function of each single mutant was additive. Evidence for an alteration in stability of the PLN/SERCA2a heterodimer was obtained in a study of double mutant N27A/N34A in which inhibitory function was regained by combining a gain of function, domain Ib mutation with a loss of function domain II mutation. These results support the proposal that PLN inhibition of SERCA2a involves, first, depolymerization of PLN and, second, the formation of inhibitory interactions between monomeric PLN and SERCA2a.

Animals↗
PubMed1998

Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.

The 31-amino acid proteolipid, sarcolipin (SLN), is associated with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1). Constructs of human and rabbit SLN and of rabbit SLN with the FLAG epitope at its N terminus (NF-SLN) or its C terminus (SLN-FC) were coexpressed with SERCA1 in HEK-293 T-cells. Immunohistochemistry was used to demonstrate colocalization of NF-SLN and SERCA1 in the endoplasmic reticulum membrane and to demonstrate the cytosolic orientation of the N terminus of SLN. Coexpression of native rabbit SLN or NF-SLN with SERCA1 decreased the apparent affinity of SERCA1 for Ca2+ but stimulated maximal Ca2+ uptake rates (Vmax). The N terminus of SLN is not well conserved among species, and the addition of an N-terminal FLAG epitope did not alter SLN function. Anti-FLAG antibody reversed both the inhibition of Ca2+ uptake by NF-SLN at low Ca2+ concentrations and the stimulatory effect of NF-SLN on Vmax. Addition of the FLAG epitope to the highly conserved C terminus decreased the apparent affinity of SERCA1 for Ca2+ relative to native SLN and decreased Vmax significantly. Mutations in the C-terminal domain showed that this sequence is critical for SLN function. Mutational analysis of the transmembrane helix, together with the additive regulatory effects of coexpression of both SLN and phospholamban (PLN) with SERCA1, provided evidence for different mechanisms of interaction of SLN and PLN with SERCA molecules. Ca2+ uptake rates in sarcoplasmic reticulum vesicles, isolated from rabbit fast-twitch muscle (tibialis anterior) subjected to chronic low frequency stimulation, were reduced by approximately 40% in 3- and 4-day stimulated muscle, with a marginal increase in apparent affinity of SERCA1 for Ca2+. SERCA1 mRNA and protein levels were unaltered after stimulation. In contrast, SLN mRNA was decreased by 15%, and SLN protein was reduced by 40%. Reduced SLN expression could explain the decrease in SERCA1 activity observed in these muscles and might represent an early functional adaptation to chronic low frequency stimulation.

Amino Acid Sequence↗
PubMed1998

Phospholamban domain I/cytochrome b5 transmembrane sequence chimeras do not inhibit SERCA2a.

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b5 were coexpressed with the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four-base circuit.

Animals↗
PubMed1998

A 37-amino acid sequence in the skeletal muscle ryanodine receptor interacts with the cytoplasmic loop between domains II and III in the skeletal muscle dihydropyridine receptor.

Interactions between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the loop linking domains II and III (II-III loop) of the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR) are critical for excitation-contraction coupling in skeletal muscle. The DHPR II-III loop was fused to glutathione S-transferase- or His-peptide and used as a protein affinity column for 35S-labeled in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR II-III loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. The use of chimeras between RyR1 and RyR2 localized the interaction to 37 amino acids, Arg1076-Asp1112, in RyR1. The RyR1 922-1112 fragment did not bind to the cardiac DHPR II-III loop but did bind to the skeletal muscle Na+ channel II-III loop. The skeletal DHPR II-III loop double mutant K677E/K682E lost most of its capacity to interact with RyR1, suggesting that two positively charged residues are important in the interaction between RyR and DHPR.

Amino Acid Sequence↗
PubMed1998

Ion tamers.

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Animals↗
PubMed1998

Bayesian modeling of muscle biopsy contracture testing for malignant hyperthermia susceptibility.

BACKGROUND: Phenotyping malignant hyperthermia (MH) by contracture testing has a low but quantifiable degree of inaccuracy, measured by its sensitivity and specificity. Quantifying the limitations inherent in diagnostic testing for MH can help resolve issues in clinical practice, such as the interpretation of a negative test and the apparent lack of complete genetic linkage to RYR1. METHODS: Bayesian models, mathematical descriptions of the outcome of diagnostic testing, were constructed. The inputs to the model include patient factors, summarized in a single number called pretest probability (PTP), and sensitivity and specificity that specify the accuracy of the entire test process. The outputs of the model include positive predictive value (PPV) and negative predictive value (NPV), which are numeric expressions of diagnostic certainty of positive and negative test results. A special case was constructed for equivocal results. RESULTS: The PPV, NPV, and efficiency of contracture testing for MH are functions of PTP, sensitivity, and specificity. The NPV is high for all clinical PTP, whereas PPV is clinically useful for moderate to high PTP. CONCLUSIONS: Diagnostic contracture testing for MH is clinically useful because of high NPV and can exclude MH with near certainty. For MH probands, the clinical grading scale for MH may guide PTP estimation, whereas for relatives of probands, PTP is a function of kinship to a known MH-susceptible relative. A sequential testing strategy optimizes diagnostic information by maximizing PTP within a pedigree. Incomplete testing of parents of an MH susceptible child can pose a significant risk of false-negative results for the untested parent. Even with optimal pedigree testing strategies, the PPV drift effect results in a considerable source of phenotypic uncertainty for genetic linkage studies.

Bayes Theorem↗
PubMed1998

Functional characterization of alternatively spliced human SERCA3 transcripts.

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.

Alternative Splicing↗
PubMed1998

Structure-function relationships in the Ca(2+)-binding and translocation domain of SERCA1: physiological correlates in Brody disease.

Alanine-scanning mutagenesis of all amino acids in transmembrane helices M4, M5, M6 and M8, which contain known Ca2+ binding residues in the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, revealed patches of mutation-sensitivity in M4, M5 and M6, but in M8. A six-residue motif, (E/D)GLPA(T/V), in M4 and M6 and its counterpart in M5 were highlighted by mutagenesis. Site-directed disulfide mapping of helices M4 and M6 demonstrated that these transmembrane helices associate as a right-handed coiled-coil. This structural information, combined with the earlier analysis of the association of each Ca2+ binding residue with either Ca2+ binding site I or site II, permitted the development of a "side-by-side" model for the two Ca2+ binding sites in the Ca(2+)-ATPase. In about half of Brody disease families, mutations create stop codons which delete all or part of the Ca2+ binding and translocation domain, resulting in loss of SERCA1 function and muscle disease.

Amino Acid Sequence↗