title: 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.
abstract: 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 35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 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 Lys954-Asp1112 retained full binding activity, whereas Leu922-Phe1075 had no binding activity. The RyR1 sequence Arg1076-Asp1112, 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, Lys954-Asp1112. These data suggest that the site of DHPR III-IV loop interaction contains elements from both the Lys954-Phe1075 and Arg1076-Asp1112 fragments. The presence of 4 +/- 0.4 microM GST-DHPR II-III or 5 +/- 0.1 microM His-peptide-DHPR III-IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922-Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922-Asp1112 binding to GST-DHPR II-III and GST-DHPR III-IV by His10-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 Asp1112.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario M5G 1L6 and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Leong
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D003593:Cytoplasm / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel