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. 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. Malignant hyperthermia (MH) 1The abbreviations used are: MH, malignant hyperthermia; CCD, central core disease; ER, endoplasmic reticulum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; ELISA, enzyme-linked immunoabsorbent assay; PBS, phosphate-buffered saline; GFP, green fluorescent protein; RyR, ryanodine receptor; IP3, inositol 1,4,5-trisphosphate. is an autosomal dominant muscle disorder in which genetically susceptible individuals among populations of humans and domestic animals respond to the administration of potent inhalational anesthetics and depolarizing skeletal muscle relaxants with high fever and skeletal muscle rigidity (1Shy G.M. Magee K.R. Brain. 1956; 79: 610-612Crossref PubMed Scopus (305) Google Scholar, 2Denborough M.A. Dennett X. Anderson R.M. Br. Med. J. 1973; 1: 272-273Crossref PubMed Scopus (122) Google Scholar). Central core disease (CCD) is a rare, non-progressive myopathy, presenting in infancy and characterized by hypotonia and proximal muscle weakness (1Shy G.M. Magee K.R. Brain. 1956; 79: 610-612Crossref PubMed Scopus (305) Google Scholar). An important feature of CCD is its close association with MH susceptibility (2Denborough M.A. Dennett X. Anderson R.M. Br. Med. J. 1973; 1: 272-273Crossref PubMed Scopus (122) Google Scholar). Although diagnosis of CCD is made on the basis of the lack of oxidative enzymatic activity in central regions of skeletal muscle fibers (3Dubowitz V. Pearse A.G.E. Lancet. 1960; : 23-24Abstract PubMed Scopus (79) Google Scholar), the diagnostic test for MH susceptibility in humans is the North American caffeine halothane contracture test (4Larach M.G. Anesth. Analg. 1989; 69: 511-515Crossref PubMed Google Scholar) or its European counterpart, the in vitro contracture test (5Group E.M. Br. J. Anaesth. 1984; 56: 1267-1271Abstract Full Text PDF PubMed Scopus (482) Google Scholar). These tests are based on the hypersensitivity of contracture of muscle strips, obtained by biopsy, to caffeine or halothane. Genetic and biochemical data have supported the view that mutations in the gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RYR1) are a major cause of MH in swine and humans (6MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (419) Google Scholar, 7MacLennan D.H. Phillips M.S. Soc. Gen. Physiol. Ser. 1995; 50: 89-100Crossref PubMed Scopus (12) Google Scholar, 8MacLennan D.H. Curr. Opin. Neurol. 1995; 8: 397-401Crossref PubMed Scopus (40) Google Scholar, 9MacLennan D.H. Philips M.S. Zhang Y. Schultz S.G. Molecular Biology of Membrane Transport Disorders. Plenum Publishing Corp., New York1996: 181-200Crossref Google Scholar, 10Loke J. MacLennan D.H. Am. J. Med. 1998; 104: 470-486Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In humans, 12 RYR1 mutations at 10 locations (C35R, G248R, G341R, R552W, R614C, R614L, R2163C, V2168M, T2206M, G2435R, R2458C, and R2458H) have been linked to MH and mutations at locations and have been linked to CCD MH PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Br. J. Anaesth. 79: Full Text PDF PubMed Scopus Google Scholar, J. J. MacLennan D.H. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. Med. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, V. J. Am. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M.S. MacLennan D.H. PubMed Scopus Google Scholar, Y. Phillips M.S. MacLennan D.H. PubMed Scopus Google Scholar). MH and CCD mutations are in in the and and and The that MH and CCD mutations are in the gene that might in a of of D.H. Phillips M.S. Soc. Gen. Physiol. Ser. 1995; 50: 89-100Crossref PubMed Scopus (12) Google Scholar, 9MacLennan D.H. Philips M.S. Zhang Y. Schultz S.G. Molecular Biology of Membrane Transport Disorders. Plenum Publishing Corp., New York1996: 181-200Crossref Google Scholar, 10Loke J. MacLennan D.H. Am. J. Med. 1998; 104: 470-486Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). were observed in of caffeine and halothane MH and CCD mutants were expressed in in HEK-293 cells J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). is not are in Ca2+ concentrations in muscle fibers of MH and CCD PubMed Scopus Google Scholar, S.G. 1989; PubMed Scopus Google Scholar, J. Google Scholar, Malignant Genetic Membrane Scholar). in and of Ca2+ concentrations in MH or CCD muscle fibers the that in muscle cells and the used by have the have to by MH and CCD mutants into HEK-293 which have a and a fluorescent to Ca2+ concentrations in cells. In an J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar), that MH or CCD mutant expressed in HEK-293 cells were more sensitive to caffeine and halothane than wild type RyR1. In the response observed for the mutant expressed in HEK-293 cells correlated with the response observed in contracture tests of muscle humans the mutations J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In MH or CCD is In the have measured Ca2+ concentrations in HEK-293 cells expressing the the caffeine-induced Ca2+ release and Ca2+ for an in sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b as a compensatory in cells expressing wild type or mutant RyR1, the caffeine of heterotetrameric MH or CCD mutants expressed in the or of and the of extracellular on caffeine of MH or CCD mutant for were obtained New and were J. Full Text PDF PubMed Google Scholar) was a of and were Molecular was was and were was obtained were of The and of the full-length rabbit skeletal muscle ryanodine (RYR1) were J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Zhang MacLennan D.H. J. Full Text PDF PubMed Scopus Google Scholar). The and of MH/CCD mutant RYR1 were J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). and of HEK-293 cells the of and PubMed Scopus Google Scholar) were as J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of were used to cells were in the with or with Ca2+ and Ca2+ were used to in HEK-293 cells as J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). was used in to caffeine-induced in Ca2+ release the ryanodine proteins expressed in HEK-293 cells. were and to the maximal Ca2+ release response observed at 10 for the caffeine and the halothane for Ca2+ were the as for the Ca2+ that the cells were with for and the was into a (CCD) of a were with The cells were to to J. Full Text PDF PubMed Scopus Google The of was obtained 10 was to be and the was to be of was used for the Ca2+ to J. Full Text PDF PubMed Scopus Google Scholar). were and for Ca2+ and data were as MacLennan D.H. J. Full Text PDF PubMed Google Scholar). of of the were by ELISA, as MacLennan D.H. PubMed Scopus Google Scholar). was used to in HEK-293 cells with RYR1 were on were for in at with PBS, and with in for 10 Cells were then with in for with PBS, by in for and with were in were on in obtained Cells were observed and with a with and a confocal were on a and data are expressed as was An test was used for of of was to In used Ca2+ and Ca2+ to the Ca2+ release of a of RyR1 mutants expressed in HEK-293 cells. Ca2+ which is and of the used to Ca2+ and was used to at to in cells in response to used Ca2+ to caffeine-induced Ca2+ release in HEK-293 cells transiently cDNA, cells cells. Ca2+ that Ca2+ release is a in or HEK-293 cells which is in the a of or more cells is In of of cells to 10 mm Ca2+ concentrations to an of and a response of In wild type or MH/CCD of higher was to 10 mm a higher than cells in Ca2+ were and an of was to be Resting Ca2+ concentrations and 10 mm caffeine-induced Ca2+ release for wild type and MH/CCD mutant RyR1 proteins were measured in cells. The Ca2+ in HEK-293 cells was with wild type RyR1 the Ca2+ to The Ca2+ in HEK-293 cells with of the 10 MH mutants R2163C, G2435R, R2458C, and R2458H) the Ca2+ to to with an of were in wild type and of the MH mutant The Ca2+ for of the CCD mutants and was higher than for of the 10 MH mutants and the Ca2+ for the CCD was significantly higher than the for the 10 MH Ca2+ concentrations for CCD mutants were with wild type were for of the CCD mutants and for and for caffeine-induced Ca2+ release and maximal for MH mutants were and and the for CCD mutants were and The caffeine-induced Ca2+ release for MH mutants was not significantly the for CCD be that MH and CCD mutants a significantly lower response to measured by Ca2+ and Ca2+ than wild type RYR1 were significantly lower for the and mutants which higher than wild type RyR1. These are with the view that CCD mutants might be more than MH for higher and lower and that MH mutants might be more than wild for lower concentrations of Ca2+ in test Ca2+ concentrations might caffeine and maximal caffeine-induced Ca2+ release, a was Resting Ca2+ concentrations with caffeine or with maximal caffeine-induced Ca2+ release a was observed and maximal caffeine-induced Ca2+ release and and maximal data not was maximal Ca2+ release for MH/CCD mutants obtained in and caffeine-induced muscle or caffeine obtained in vitro contracture test in an V. J. Am. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). was observed maximal caffeine-induced Ca2+ release and caffeine-induced muscle and maximal caffeine-induced Ca2+ release and caffeine test Ca2+ were lower in MH/CCD and were used to Ca2+ release, by of the of Ca2+ Ca2+ by of sarco(endo)plasmic reticulum Ca2+-ATPase Ca2+ release, the of the Ca2+ PubMed Scopus Google Scholar). Ca2+ by of the an endogenous in HEK-293 in of and of the X. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). low Ca2+ respond with low Ca2+ release the of of that was a close for Ca2+ release by of in RYR1 cells. These cells were by 10 mm caffeine and In cells with MH/CCD mutant to homotetrameric or heterotetrameric caffeine-induced Ca2+ release was correlated with thapsigargin-induced Ca2+ release close among and thapsigargin-induced Ca2+ release that maximal caffeine-induced Ca2+ release the size of the Ca2+ store. The lower maximal caffeine-induced Ca2+ release in MH/CCD mutants is to be to a lower Ca2+ in is to Ca2+ MH/CCD mutant Ca2+ among and RYR1 Ca2+ release was measured by Ca2+ and was as an of the size of the Ca2+ store. that Ca2+ release in HEK-293 cells was than Ca2+ release in cells with wild was higher than Ca2+ release in cells with CCD or MH mutant The Ca2+ release in cells with MH/CCD mutants the view that mutants are more than wild type RyR1. The of the Ca2+ by of wild type RyR1 to test the that cells with that release Ca2+ endogenous in an to Ca2+ by Ca2+ concentrations to cells with wild type or mutant RYR1 the capacity to a Ca2+ were measured by and was measured in cells When HEK-293 cells were with wild type the content was increased to or with MH mutant to an in content to or with CCD mutant to an in content to or Although for is the in in cells be a of and confocal were used in a of the of content in cells with mutant RyR1. and are green fluorescent and rabbit RyR1 mutant were into HEK-293 cells in a in cells was as an of cells. of rabbit RyR1, by that of cells expressed and RyR1, of cells expressed and of cells expressed RyR1 The which were was increased by with cells supporting that the content of cells was These that in the of enhanced in RYR1 or MH/CCD mutant cells. The maximal caffeine-induced Ca2+ release for MH/CCD mutant homotetrameric was than for wild type is in to which that muscle fibers malignant individuals more in low concentrations of caffeine and halothane than fibers in with enhanced Ca2+ Although homotetrameric MH/CCD mutant expressed in HEK-293 cells have been to be more sensitive to caffeine and halothane than wild type RyR1 J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar), heterotetrameric MH/CCD mutant are in In to heterotetrameric wild type and mutant RyR1 be and were in a with wild type When HEK-293 cells expressed heterotetrameric caffeine were to be of cells expressing homotetrameric or and cells expressing homotetrameric wild type RYR1 and 10 mm caffeine-induced Ca2+ release in heterotetrameric MH/CCD mutants were higher than in homotetrameric MH/CCD suggesting that are than is that the activity of is with the activity of Ca2+ release in skeletal muscle to Ca2+ the of that observed in cells expressing MH/CCD mutants be as a compensatory enhanced endogenous was not to for the higher Ca2+ concentrations to mutant or is that compensatory in cells the and to at a more by with SERCA1 high of activity are observed MacLennan D.H. PubMed Scopus Google Scholar). SERCA1 was increased the of Ca2+ the caffeine-induced Ca2+ release and the of Ca2+ caffeine of cells with wild type or MH/CCD mutant RyR1 of mutants or with SERCA1 Ca2+ concentrations The of SERCA1 with or with wild type RyR1, or with or with wild type RyR1 caffeine increased and increased maximal Ca2+ release for RyR1 mutants These that SERCA1 Ca2+ and Ca2+ that increased the mutant in the ER, Ca2+ is in an in used Ca2+ to test caffeine-induced Ca2+ release in MH/CCD mutant that the heterotetrameric mutant SERCA1 and the heterotetrameric mutant SERCA1 were significantly more sensitive to low concentrations of caffeine and and halothane and than wild type RyR1 SERCA1 The MH/CCD mutant SERCA1 more Ca2+ by low concentrations of caffeine and halothane than wild type RyR1 SERCA1 of the were obtained HEK-293 cells in a mm that caffeine-induced Ca2+ release cells was by Ca2+release Ca2+ caffeine-induced Ca2+ release in cells was measured in Ca2+-free Ca2+ release, by caffeine or and measured by in was and Ca2+ concentrations in and cells were lower in a Ca2+-free than in These that extracellular Ca2+ a on Ca2+ concentration. In that caffeine-induced Ca2+release be by the of in indicating that caffeine-induced Ca2+ release depends on the Ca2+ store. In RYR1 cells were in Ca2+-free with for and caffeine-induced Ca2+release was measured in Ca2+-free medium, then in medium, in Ca2+-free In Ca2+-free medium, caffeine were the caffeine In caffeine of Ca2+ release be The of mm in an in Ca2+ and an in the amplitude of caffeine-induced Ca2+release When the cells were to Ca2+-free medium, the caffeine response was were obtained for in cells These that extracellular Ca2+ concentrations have a on Ca2+ When measured caffeine for wild type and and mutants in Ca2+-free medium, observed that the mutant were more sensitive than wild type to caffeine as were in The caffeine for RyR1, and in Ca2+-free were and These are to higher than the measured in The lower caffeine in Ca2+-free have been to the lower Ca2+ in Ca2+-free suggesting that Ca2+ concentrations the caffeine of RyR1. In a J. 1998; PubMed Scopus Google Scholar) that an isoform of the ryanodine is expressed in HEK-293 cells. The of endogenous measured was be sensitive of cells or by of not proteins in of HEK-293 cells by observed of J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Although J. 1998; PubMed Scopus Google Scholar) a for was not J. 1998; PubMed Scopus Google Scholar) in Ca2+ of in of HEK-293 cells by the of high of endogenous activity were in have by Ca2+ measured the at cells in J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). measured a in the in HEK-293 measured a in HEK-293 cells with wild type RyR1 that was than In in which of HEK-293 measured the of endogenous Ca2+ release in and cells. that of cells to 10 mm to an of and response at In wild type or mutant of cells to a higher than Ca2+ concentrations The in the of and of J. 1998; PubMed Scopus Google Scholar) are to be to the in which were J. 1998; PubMed Scopus Google Scholar) not caffeine-induced Ca2+ release to the cells of In to Ca2+ release, to and then and to mm at high caffeine to the endogenous the of Ca2+ release was than that which were in the of J. 1998; PubMed Scopus Google Scholar) that for the Ca2+ release to endogenous RyR, not be to used a of Ca2+ release in cells. In D.H. Phillips M.S. Soc. Gen. Physiol. Ser. 1995; 50: 89-100Crossref PubMed Scopus (12) Google Scholar, 9MacLennan D.H. Philips M.S. Zhang Y. Schultz S.G. Molecular Biology of Membrane Transport Disorders. Plenum Publishing Corp., New York1996: 181-200Crossref Google Scholar, 10Loke J. MacLennan D.H. Am. J. Med. 1998; 104: 470-486Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), that might be Ca2+ MH mutant that compensatory to of Ca2+ muscle in MH swine might Ca2+ release an Ca2+ release channel to muscle that CCD mutations might to more Ca2+ release, which Ca2+ in the core of the not in the of Ca2+ be by or in the D.H. Philips M.S. Zhang Y. Schultz S.G. Molecular Biology of Membrane Transport Disorders. Plenum Publishing Corp., New York1996: 181-200Crossref Google J. MacLennan D.H. Am. J. Med. 1998; 104: 470-486Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In observed higher in HEK-293 cells with wild suggesting that the expressed channel might the of the Ca2+ store. might the that have in to a HEK-293 expressing When the cells were with RYR1 MH Ca2+ concentrations were that of wild type cells in were not significantly wild was the that the Ca2+ was significantly wild type and MH mutant RyR1 proteins for the CCD mutant RyR1 proteins expressed in HEK-293 cells. the CCD and Ca2+ concentrations were significantly wild type RyR1. caffeine-induced Ca2+ release, measured by Ca2+ and Ca2+ was lower in cells with MH/CCD mutants than with wild measured the size of the Ca2+ that was by as which Ca2+ which of Ca2+ and which Ca2+ the The close of and thapsigargin-induced Ca2+release that the maximal caffeine-induced Ca2+release is to the size of the Ca2+ store. a in amplitude of Ca2+ release in cells to the and was not by cells a in response to and is more that was based on as or among were to of of Ca2+ in response to an RyR1 be to lower the for caffeine-induced Ca2+ release at the to as observed in These that CCD and MH mutant are more than wild Resting Ca2+ concentrations were not correlated with caffeine or with maximal caffeine-induced Ca2+ lack of is with and with J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar), which in caffeine and halothane MH and CCD The that not have a major on the caffeine of MH/CCD for Ca2+ release MH/CCD mutant proteins were correlated with maximal caffeine and the maximal caffeine-induced Ca2+ release was correlated with caffeine indicating that higher Ca2+ caffeine In with rabbit an in Ca2+ to been to channel activity J. Science. PubMed Scopus Google Scholar, J. J. Full Text PDF PubMed Scopus Google Scholar, J. Full Text PDF PubMed Scopus Google Scholar). high Ca2+ channel caffeine The is that low concentrations caffeine the lower Ca2+ in the sarcoplasmic reticulum a compensatory in MH/CCD skeletal muscle cells. halothane an MH not on the of MH/CCD mutants to halothane on the the of Ca2+ and the Ca2+ the sarcoplasmic individuals MH mutations not higher to caffeine and halothane. The of lower caffeine-induced Ca2+ release in HEK-293 cells with MH/CCD mutants is not with in MH individuals have MH/CCD and an in the of for enhanced Ca2+ MH/CCD mutants higher caffeine-induced Ca2+ release were with wild type RyR1 and SERCA1. The caffeine of the heterotetrameric MH/CCD mutants was that of the homotetrameric MH/CCD mutants and wild type RyR1 maximal in MH/CCD heterotetrameric mutants were higher than in homotetrameric MH/CCD RyR1 for the MH for the of Ca2+ release and the for Anesth. Analg. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. 1992; Full Text PDF PubMed Google Scholar, J. 50: Google Scholar, 1995; PubMed Scopus Google Scholar). Ca2+ release were in heterotetrameric MH/CCD mutants and wild type the heterotetrameric MH/CCD mutants SERCA1 were more sensitive to low concentrations of caffeine and halothane than the cells with wild type RyR1 SERCA1 that of SERCA1 the Ca2+ and that of heterotetrameric MH/CCD mutants the of homotetrameric MH/CCD mutant the of wild MH, and CCD mutant RyR1 in HEK-293 cells be as a of The of wild type RyR1 the Ca2+ the size of the Ca2+ and content and activity These be on the basis of an increased of the Ca2+ which is for by of with a of the Ca2+ store. at of the enhanced of Ca2+ the in a higher Ca2+ concentration. of MH or CCD mutant RyR1 and of to a higher for Ca2+ was not the for MH and CCD mutants were to be that the to Ca2+ for MH and CCD mutants was the more higher of In to be SERCA1 with and mutant that of higher of SERCA1 not Ca2+ concentrations Ca2+ for RyR1 These into the in which in proteins and are is a more Ca2+ proteins in the sarcoplasmic the and D.H. Phillips M.S. Soc. 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PubMed Scopus Google Scholar). that is enhanced in HEK-293 cells and that the CCD mutant channel the of endogenous The higher and of Ca2+ was not observed higher of for the MH and CCD mutants than for wild type RyR1. the that were made a for to be of the of CCD muscle is a of the in the core 1989; PubMed Scopus Google Scholar). The of a of or that compensatory a to In the Ca2+ that have observed in of the size of the Ca2+ and Ca2+ concentrations in cells with wild MH, or CCD mutant RyR1. The of wild type RyR1 Ca2+ In response to increased Ca2+ the cells the to more is as a higher Ca2+ for the cells with wild type RyR1. The MH mutant is more than wild type RyR1, in an Ca2+ and enhanced the for an increased is not Ca2+ of the is higher than Ca2+ into the store. lower Ca2+ the the CCD more in higher Ca2+ and an higher Ca2+ are not the Ca2+ is The is that the cells with a CCD mutant have the and the Ca2+ store. These the view that MH and CCD mutants in a of muscle by Ca2+ to muscle by to the core of the muscle compensatory is D.H. Phillips M.S. Soc. Gen. Physiol. Ser. 1995; 50: 89-100Crossref PubMed Scopus (12) Google Scholar, 9MacLennan D.H. Philips M.S. Zhang Y. Schultz S.G. Molecular Biology of Membrane Transport Disorders. Plenum Publishing Corp., New York1996: 181-200Crossref Google Scholar, 10Loke J. MacLennan D.H. Am. J. Med. 1998; 104: 470-486Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Ca2+ concentrations were not caffeine or halothane among wild MH, and CCD mutant RyR1 were of mutant a the size of the Ca2+ was the of that extracellular Ca2+ a on caffeine-induced Ca2+ release and Ca2+ is to an RyR1 caffeine in a Ca2+-free medium, Ca2+ is to extracellular is the caffeine measured in Ca2+-free not the of the RyR1 channel the size of the Ca2+ in the cells. The lower caffeine in Ca2+-free than in that Ca2+ concentrations RyR1 caffeine In have at that the RyR1 caffeine These are the of RyR1 the size of the Ca2+ and the Ca2+ concentration. have that MH mutants are more sensitive to a variety of channel and than wild type D.H. Philips M.S. Zhang Y. Schultz S.G. Molecular Biology of Membrane Transport Disorders. Plenum Publishing Corp., New York1996: 181-200Crossref Google Scholar, J. MacLennan D.H. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Full Text PDF PubMed Scopus Google Scholar, Anesth. Analg. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. 1992; Full Text PDF PubMed Google Scholar, J. 50: Google Scholar, 1995; PubMed Scopus Google Scholar) and are sensitive to Am. J. Physiol. PubMed Google Scholar). the cause of MH is the hypersensitivity of RyR1 mutant proteins to the of MH in humans be by the size of the Ca2+ and Ca2+ which be by a of proteins in skeletal The were obtained with HEK-293 cells with wild type and MH/CCD mutant RYR1 and the data were obtained cells skeletal muscle cells in that lack of the proteins that Ca2+ The of the HEK-293 is that the of and MH/CCD mutants in a the of and which not be in skeletal muscle is or to of MH mutant and are is more to in RyR1 MH/CCD and might be to be to the compensatory of to skeletal muscle cells. The of Ca2+ in HEK-293 cells is a that be the of of the Ca2+ release be of be to the compensatory that are in transiently HEK-293 cells. the in the is to that MH/CCD which are more in HEK-293 are more in skeletal muscle cells.