The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis.
Rapid Ca2+ efflux from intracellular stores during cardiac muscle excitation-contraction coupling is mediated by the ryanodine-sensitive calcium-release channel, a large homotetrameric complex present in the sarcoplasmic reticulum. We report here the identification, primary structure and topological analysis of the ryanodine receptor-calcium release channel from human cardiac muscle (hRyR-2). Consistent with sedimentation and immunoblotting studies on the hRyR-2 protein, sequence analysis of ten overlapping cDNA clones reveals an open reading frame of 14901 nucleotides encoding a protein of 4967 amino acid residues with a predicted molecular mass of 564 569 Da for hRyR-2. In-frame insertions corresponding to eight and ten amino acid residues were found in two of the ten cDNAs isolated, suggesting that novel, alternatively spliced transcripts of the hRyR-2 gene might exist. Six hydrophobic stretches, which are present within the hRyR-2 C-terminal 500 amino acids and are conserved in all RyR sequences, may be involved in forming the transmembrane domain that constitutes the Ca(2+)-conducting pathway, in agreement with competitive ELISA studies with a RyR-2-specific antibody. Sequence alignment of hRyR-2 with other RyR isoforms indicates a high level of overall identity within the RyR family, with the exception of two important regions that exhibit substantial variability. Phylogenetic analysis suggests that the RyR-2 isoform diverged from a single ancestral gene before the RyR-1 and RyR-3 isoforms to form a distinct branch of the RyR family tree.
Tunwell
R E
RE
MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, U.K.
Wickenden
C
C
Bertrand
B M
BM
Shevchenko
V I
VI
Walsh
M B
MB
Allen
P D
PD
Lai
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003062:Codon; D018076:DNA, Complementary; D004330:Drosophila; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D015183:Restriction Mapping; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D000906:Antibodies; D015220:Calcium Channels; D003062:Codon; D018076:DNA, Complementary; D009124:Muscle Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium