IDENTIFICATION OF A 2 EF-HAND CA2-MUSCLE RYANODINE RECEPTOR( BINDING DOMAIN IN LOBSTER SKELETAL)CA2+ RELEASE CHANNEL/

The lobster skeletal muscle Ca2+ release channel, known also as the ry anodine receptor, is composed of four polypeptides of similar to 5000 amino acids each, like its mammalian counterparts. Clones encoding the carboxy-terminal region of the lobster ryanodine receptor were isolat ed from a lobster skeletal muscle cDNA library. Analysis of the deduce d 1513 carboxy-terminal amino acid sequence suggests a cytoplasmic Ca2 + binding domain consisting of two EF-hand Ca2+ binding motifs (amino acid residues 594-656). The Ca2+ binding properties of this domain wer e assessed by preparing bacterial fusion proteins with sequences from the lobster Ca2+ binding domain and the corresponding sequences of the rabbit cardiac and skeletal muscle ryanodine receptors. The lobster s keletal muscle fusion protein bound Ca-45(2+) in Ca2+ overlays, and bo und two Ca2+ under equilibrium binding conditions with a Hill dissocia tion constant (K-H) of 0.9 mM and coefficient (n(H)) of 1.4. Rabbit sk eletal and cardiac fusion proteins bound two Ca2+ with K(H)s of 3.7 an d 3.8 mM and n(H)s of 1.1 and 1.3, respectively. Similar to results pr eviously reported for the mammalian RyRs, the lobster RyR was activate d by micromolar Ca2+ and inhibited by millimolar Ca2+, as determined i n single-channel and [H-3]ryanodine binding measurements. These result s suggest that the two EF-hand Ca2+ binding domain of the lobster Ca2 release channel as well as the corresponding regions of the mammalian channels may play a role in Ca2+ inactivation of sarcoplasmic reticul um Ca2+ release.