IMAGAWA Toshiaki National Cardiovascular Center Research Institute Department of Molecular Physio, 循環分子生理部, 室長 (20142177)
NAKAMURA Hiroshi National Cardiovascular Center Research Institute Department of Molecular Physio, 循環分子生理部, 室長 (30029508)
中崎 育明 国立循環器病センター研究所, 循環分子生理部, レジデント
NAKASAKI Yasuaki National Cardiovascular Center Research Institute Department of Molecular Physio
|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1991 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1990 : ¥1,300,000 (Direct Cost : ¥1,300,000)
1)The sequence of 4968(or 4976 with an insertion)amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by-cloning and sequencing the CDNA. This protein is homologous in antino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with MRNA derived from the cardiac ryanodine receptor CDNA exhibit Ca^<2+>-dependent Cl^- current in response caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor MRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac MRNA. Ibis results, in conjunction with cloning and analysis of partial CDNA sequence, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.
2)The exogenous addition of tie catalytic subunit of cAMP-depeiident protein kinase(PK
A), cOMP-dependent protein kinase(PKG), or calmodulin(CaM)induced rapid, phosphorylation of the ryanodine receptor(Ca^<2+> release channel)in canine cardiac microsomes treated with lmM[gamma- ^<32>P]ATP. Added pro(ein kinase C(PKC)also phosphorylated the cardiac ryanodiiie receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependen(phosphorylation of the ryanodine receptor was comparable to the maximum level of[ ^3H]ryanodine binding in cardiac microsomes. wherets the level of CaM-dependent phosphorylation was about 4 times greater. Phospliorylation by PKA. PKG, and PKC increased[ ^3H]rryanodine binding in cardiac microsomes by 22<plus-minus>5, 17<plus-minus>4, and 15<plus-minus>9 %(average <plus-minus>SD, n=4-5), respectively. In contrast, incubation of microsomes with 5 mu M CaM alone and 5 mu M CaM plus 1 mM ATP decreased[ ^3H]ryanodine binding by 38<plus-minus>14 and 53<plus-minus>15 %(average <plus-minus>SD. n=6). respectively. Pliosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA. FIKO. and PKC predominantly phosphorylate serine residue(s)in the same phospliopeptide(peptide I). whereas the endogetious CaM-kinase phosphorylates serine residue(s)in a different phosphopeptide(peptide 4). Photoaffinity labeling or microsomes with photoreactive ^<125>I-labeled CaM revealed that CaM bound to a high niolecular weight protein, which was immunoprecipitated by a monoclogial antibody against the cardiac ryanodine receptor. These results suggest that protein kittase-det, eiidciit pliospliorylatioii and CaM play important regulatory roles iii tlie function of the cardiac sarcoplasniic reticulum Ca^<2+> release channel.
3)We constructed an expression plasmid(pMAMCRR51)that carries the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the mouse mammary tumor virus promotor and E. coli xanthine-guanine phosphoribosyltransferase(gpt)as a selectable marker. Chinese hamster ovary(CHO)cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca^<2+> transients were. selected. Immunoprecipitation with a monocional antibody against the canine cardiac ryatiodine receptor revealed that the cell clones thus selected exhibited Ca^<2+>-dependent[ ^33H]ryanodine binding activity, which was stimulated by 10 mM ATP or 1 M KCI. The apparent dissociation constatit(Kd)for [ ^3H]ryanodine was 5.2 nM. which was similar to the Kd observed with cardiac microsomes. Immunoprecipitation also demonstrated that the cell clones produced a protein indistinguishable in Mr from the ryatiodine receptor in canine cardiac microsomes. The amount of ryanodine receptor expressed in CHO cells increased significantly after dexamethasone induction. We examined the effect of Ca^<2+> and caffeine on Ca^<2+> release from intracellular Ca^<2+> stores using saponin-skinned CHO cells. Addition of micromolar levels of Ca^<2+> evoked rapid Ca^<2+> release from skinned CHO cells expressing the cardiac ryanodine receptor. In the presence of 0.1 muM Ca^<2+>, however, some Ca^<2+> release observed with the skinned CHO cells, but addition of 5 mM caffeitie stimulated Ca^<2+> release. The amounl of Ca^<2+> release induced by addition of micromolar levels of Ca^<2+> or 5 muM caffeine was similar to that observed after addition of ionomycin. These results clearly demonstrate that the cardiac ryanoditie receptor expressed in CHO cells functions as a Ca^<2+>-induced Ca^<2+> release channel.