Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle...

Major progress has been made in elucidating the calcium release mechanism involved in excitation-contraction coupling. The ryanodine receptor of sarcoplasmic reticulum has been isolated and found to be morphologically identical to the foot structure, which is involved in the junctional association of terminal cisternae with the transverse tubule. The foot structure also contains the calcium release channel itself. For this reason, we refer to the foot structure as the junctional channel complex (JCC). The JCC consists of an oligomer of a single high molecular weight protein. Although progress has been made in characterizing important aspects of the structure and function of the JCC, further understanding of the JCC protein subunit awaits the molecular cloning of the JCC. We report on the isolation of cDNA clones encoding portions of the JCC from rabbit fast-twitch skeletal muscle and its tissue distribution and expression. The large size and lack of solubility of the JCC protein posed particular challenges to cloning this molecule. Among these was the necessity to develop techniques for partially digesting the JCC protein subunit with endoproteases in the presence of detergent. With this approach we obtained partial amino acid sequences from regions of the JCC and designed oligonucleotide primers and probes to synthesize and screen cDNA libraries. The rabbit skeletal muscle JCC mRNA encodes an approximately 16-kilobase mRNA present in skeletal, heart, and aortic smooth muscle, as determined by RNA blot analysis with a 700-base-pair cDNA probe. Whereas the JCC mRNA appears to be relatively abundant in adult rabbit fast-twitch skeletal muscle, it is much less abundant in heart and smooth muscle. The JCC mRNA in BC3H1 (a myoblast cell line) is reversibly regulated by growth factors in a manner similar to muscle-specific contractile protein genes.