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Identification of a two EF-hand Ca2+ binding domain in lobster skeletal muscle ryanodine receptor/Ca2+ release channel.

The lobster skeletal muscle Ca2+ release channel, known also as the ryanodine receptor, is composed of four polypeptides of approximately 5000 amino acids each, like its mammalian counterparts. Clones encoding the carboxy-terminal region of the lobster ryanodine receptor were isolated from a lobster skeletal muscle cDNA library. Analysis of the deduced 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 were 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 skeletal muscle fusion protein bound 45Ca2+ in Ca2+ overlays, and bound two Ca2+ under equilibrium binding conditions with a Hill dissociation constant (KH) of 0.9 mM and coefficient (nH) of 1.4. Rabbit skeletal and cardiac fusion proteins bound two Ca2+ with KHs of 3.7 and 3.8 mM and nHs of 1.1 and 1.3, respectively. Similar to results previously reported for the mammalian RyRs, the lobster RyR was activated by micromolar Ca2+ and inhibited by millimolar Ca2+, as determined in single-channel and [3H]ryanodine binding measurements. These results 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 reticulum Ca2+ release.