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.
Highlights
The importance of the DHPR II–III loop in E-C coupling has been demonstrated by Tanabe et al [10, 11] through the study of dysgenic myotubes lacking DHPR
The DHPR III–IV loop may play a role in E-C coupling, and a mutation in this loop could modify the interaction between the dihydropyridine and ryanodine receptors, odine receptor; DHPR, dihydropyridine receptor; Ni2ϩ-NTA, Ni2ϩ-nitrilotriacetic acid; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin
Identification of an RyR1 Sequence Interacting with the DHPR III–IV Loop—We scanned 35S-labeled, in vitro translated RyR1 fragments for interactions with the DHPR III–IV loop immobilized on a Ni2ϩ column as a His-peptide fusion protein (Fig. 1)
Summary
The importance of the DHPR II–III loop in E-C coupling has been demonstrated by Tanabe et al [10, 11] through the study of dysgenic myotubes lacking DHPR. A protein affinity chromatography approach was used to identify an interaction site on RyR1 for the DHPR II–III loop [15]. A mutation in the DHPR III–IV loop has been linked to susceptibility to malignant hyperthermia [17]. The studies in dysgenic mice [11] that identify the DHPR II–III loop as being critical for RyR1 activation neither rule out nor implicate the III–IV loop in E-C coupling. We used protein affinity chromatography to demonstrate that the DHPR III–IV loop can interact with RyR1
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