CRYOELECTRON MICROSCOPY RESOLVES FK506-BINDING PROTEIN SITES ON THE SKELETAL-MUSCLE RYANODINE RECEPTOR

A 12-kDa immunophilin (FKBP12) is an integral component of the skeleta l muscle ryanodine receptor (RyR), The RyR is a hetero-oligomeric comp lex with structural formula (FKBP)(4)(Ryr1)(4), where Ryr1 is the 565- kDa product of the Ryr1 gene. To aid in the detection of the immunophi lin's location in the receptor, we exchanged the FKBP12 present in RyR -enriched vesicles derived from sarcoplasmic reticulum with an enginee red construct of FKBP12 fused to glutathione S-transferase and then is olated the complexes. Cryoelectron microscopy and image averaging of t he complexes (in an orientation displaying the RyR's fourfold symmetry ) revealed four symmetrically distributed, diffuse density regions tha t were located just outside the boundary defining the cytoplasmic asse mbly of the RyR. These regions are attributed to the glutathione trans ferase portion of the fusion protein because they are absent from rece ptors lacking the fusion protein. To more precisely define the locatio n of FKBP12, we similarly analyzed complexes of RyR containing FKBP12 itself. Apparently some FKBP is lost during purification or storage of the RyR because, to detect the receptor-bound immunophilin, it was ne cessary to add FKBP12 to the purified receptor before electron microsc opy. Averaged images of these complexes showed a region of density tha t had not been observed previously in images of isolated receptors, an d its position, along the edges of the transmembrane assembly, agreed with the position of the FKBP12 deduced from the experiments with the fusion protein. The proposed locations for FKBP12 are about 10 nm from the transmembrane baseplate assembly that contains the ion channel of the RyR.