Debra K. McMahon, Ph.D.

Dissertation research performed under the direction of Nadia N. Malouf

ABSTRACT
Excitation contraction coupling (ECC) in skeletal muscle involves the interaction of the dihydropyridine sensitive voltage dependent calcium channel (DHP-R) of the transverse tubule (t-tubule) and the ryanodine binding calcium release channel (RR) of the sarcoplasmic reticulum (SR). The DHP-R and the RR are preferentially localized in their respective membranes at the triad, the site of ECC in skeletal muscle. The organization and stabilization of the triad during development is imperative for the acquisition and maintenance of efficient ECC.  Recently, our laboratory has found an isoform of the DHP-R which has only two of the four domains in the previously reported DHP-R. As a first step in understanding the function of this isoform, we have (1) characterized the C$\sb2$C$\sb{12}$ mouse skeletal muscle cell line to examine its role in triad biogenesis, (2) localized the two DHP-R isoforms in sucrose gradient fractions isolated from adult and immature rabbit skeletal muscle and in intact adult and immature rabbit skeletal muscle, (3) performed binding studies with the DHP ligand PN200-110 with membrane preparations enriched in the two DHP-R isoforms, and (4) attempted (a) the expression of the cytoplasmic loop absent in the two motif DHP-R which is important in skeletal muscle type ECC, (b) the expression of the abbreviated DHP-R in Xenopus oocytes, and (c) the expression of the abbreviated DHP-R in the rabbit reticulocyte lysate system.  We have found that (1) although the C$\sb2$C$\sb{12}$ cell line has well developed sarcomeres and triads, the paucity of DHP-Rs in this cell line hampers the study of triad biogenesis using the DHP-R as a probe, (2) the two DHP-R isoforms colocalize in sucrose gradient fractions of adult and immature rabbit skeletal muscle membranes and in intact adult and immature skeletal muscle, (3) the affinity for the DHP-R in immature rabbit skeletal muscle membranes may be higher than that for the DHP-R in adult rabbit skeletal muscle membranes, and (4) the abbreviated DHP-R isoform expressed in the TnT rabbit reticulocyte lysate system has a mobility on SDS-PAGE which is consistent with the polypeptide present in skeletal muscle membranes.