Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantitie s sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and express ed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3), RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the for m of a GST fusion as the affinity ligand, Purification of GST-RyR3 was achi eved by affinity chromatography by using glutathione-Sepharose. Purified re combinant RyR3 and GST-RyR3 proteins exhibited high-affinity [H-3]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhib ition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 app eared very similar to that of the native RyR3 purified from bovine diaphrag m, Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are loca ted in the "clamp" structures that form the corners of the square-shaped cy toplasmic region of homotetrameric RyR3, This study describes the 3D recons truction of a recombinant ryanodine receptor and it demonstrates the potent ial of this technology for characterizing functional and structural perturb ations introduced by site-directed mutagenesis.