The cytoplasmic loops between domains II and III and domains III and IV in the skeletal muscle dihydropyridine receptor bind to a contiguous site in the skeletal muscle ryanodine receptor

Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR), Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization induced inactivation of RyR1. The DHPR III-IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for S-35-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1, RyR1 residues Leu(922)-Asp(1112) bound specifically to the DHPR III-IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys(954)- Asp(1112) retained full binding activity, whereas Leu(922)- Phe(1075) had no binding activity. The RyR1 sequence Arg(1076)-Asp(1112), previously shown to interact with the DHPR II-III loop (Leong, P,, and MacLennan, D,, H. (1998) J, Biol, Chem. 273, 7791-7794), bound to DHPR III-IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys(954)-Asp(1112) These data suggest that the site of DHPR III-TV loop interaction contains elements from both the Lys(954)-Phe(1075) and Arg(1076)-Asp(1112) fragments. The presence of 4 +/- 0.4 mu M GST-DHPR II-III or 5 +/- 0.1 mu M His-peptide-DHPR III-IV was required for half-maximal co-purification of S-35-labeled RyR1 Leu(922)-Asp(1112) On glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of S-35-labeled RS RI Leu(922)-Asp(1112) binding to GST-DHPR II-III and GST-DHPR III-IV by His(10)-DHPR II-III and His-peptide-DHPR III-IV was observed. These studies indicate that the DHPR II-III and III-IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys(954) and Asp(1112).