THE CYTOPLASMIC LOOPS BETWEEN DOMAIN-II AND DOMAIN-III AND DOMAIN-IIIAND DOMAIN-IV IN THE SKELETAL-MUSCLE DIHYDROPYRIDINE RECEPTOR BIND TOA 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 sarcop lasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR), Interactions b etween RyR1 and DHPR are critical for the depolarization-induced activ ation 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 (GS T) or His-peptide and used as a protein affinity column for S-35-label ed, 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 r yanodine receptor (RyR2) did not. Construction of chimeras between RyR 1 and RyR2 showed that amino acids Lys(954)- Asp(1112) retained full b inding activity, whereas Leu(922)- Phe(1075) had no binding activity. The RyR1 sequence Arg(1076)-Asp(1112), previously shown to interact wi th the DHPR II-III loop (Leong, P,, and MacLennan, D,, H. (1998) J, Bi ol, 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 in teraction contains elements from both the Lys(954)-Phe(1075) and Arg(1 076)-Asp(1112) fragments. The presence of 4 +/- 0.4 mu M GST-DHPR II-I II or 5 +/- 0.1 mu M His-peptide-DHPR III-IV was required for half-max imal co-purification of S-35-labeled RyR1 Leu(922)-Asp(1112) On glutat hione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibiti on 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-I V was observed. These studies indicate that the DHPR II-III and III-IV loops bind to contiguous and possibly overlapping sites on RyR1 betwe en Lys(954) and Asp(1112).