Abstract
Expression studies with skeletal and cardiac muscle cDNAs have suggested that the putative cytoplasmic loop region of the dihydropyridine receptor (DHPR) alpha 1 subunit between transmembrane repeats II and III (DCL) is a major determinant of the type of excitation-contraction coupling (skeletal or cardiac) in rescued dysgenic muscle cells (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569). In this study, the possibility of a direct functional interaction with the sarcoplasmic reticulum ryanodine receptor/Ca2+ release channel has been tested by expressing the DCLs of the mammalian skeletal and cardiac muscle DHPR alpha 1 subunit in Escherichia coli. The purified peptides activated the skeletal muscle ryanodine receptor/Ca2+ release channel in single channel and [3H]ryanodine binding measurements, by increasing channel open probability and the affinity of [3H]ryanodine binding, respectively. The two peptides did not activate the cardiac muscle Ca2+ release channel. Other proteins (polylysine, serum albumin) also increased [3H]ryanodine binding and Ca2+ release channel activity, but their activation mechanisms were distinguishable from DCLs. These results show that the II-III cytoplasmic loop of the skeletal and cardiac DHPR alpha 1 subunit functionally interacts with the skeletal, but not cardiac, muscle Ca2+ release channel. Furthermore, our studies suggest that in addition to the DHPR, the sarcoplasmic reticulum Ca2+ release channel may determine the type of E-C coupling that exists in muscle.
Highlights
- (DCL) is a major determinant of the type of excitation- Theskeletalandcardiac RYRs are homologouschannels contractioncoupling in rescued comprised of four M, 565,000 polypeptides (6).the dysgenic musclecells
Expression a n d Purification of DCLs-The cytoplasmic loops tion of micromolar concentrations of ryanodine modified the between transmembrane repeats I1 and I11 of the dihydropyridine receptor (DHPR) a1 K+-conductance of the SDCL-activated channels in a manner subunit (DCL) from rabbit skeletal muscle (SDCL) and rabbit highly characteristic for the skeletalCa2+release channel (191, cardiac muscle (CDCL) (Fig. lA) wereexpressed in E. coli, confirming that ryanodine-sensitive channels were realong with a n amino-terminal (12 amino acid residues) Tag corded in this study
BSA significantlyincreased the B, but not KDvalue of [3H]ryanodinebinding (Fig. 6, Table 11).The interaction of SDCL with the skeletal muscle Ca2+release channel was characterized by determining its activating effects in the presence of CDCL, BSA,and polylysine.The activating effects of 10 p~ SDCL and 1p~ polylysine (Fig.5)were additive,whereas no or only small further increases were measured in assay media containing 10 p~ CDCL or 10 p~ BSA.Taken together, the [3H]ryanodine binding studies suggested that SDCL and CDCL likely activate the skeletal muscle Ca2+release channel by a similar mechanism, but that there exist significant differences with regard to their action on the skeletal and cardiac muscle RYR isoforms
Summary
Materials-PHlRyanodine (54.7 Cilmmol) was obtainedfrom DuPont NEN. Unlabeled ryanodine was from AgriSystems International (Wind Gap, PA). Plasmid pETlld, E. coli strain BL21(DE3)and T7.Tag monoclonal antibody were from Novagen (Madison, WI). DEAE-Sephacel, hydroxyapatite, SDS-PAGE molecular weight markers, bovine serum albumin (BSA),insulin, cytochrome c, polylysine ( M , 26,3001, and polyglutamate ( M , 13,300)were purchased from Sigma, and phospholipids from Avanti (Birmingham,AL). All other chemicals were of analytical grade. Expression and Purification of SDCL and CDCL-cDNAs encoding SDCL (GhP6to Leu791)( 8 )or CDCL (Asp788 toGlnSz2)(9)were ampli-. The nucleotide sequences of cloned PCR products were determined and showed agreement with those published. The PCR products were cloned into the BamHI site of pETlld expression vector with in-frame insertion. The recombinant plasmids pETlld-sdcl or pETlld-cdcl were expressed in E. coli strain BL21(DE3)a s described elsewhere (14).
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