Ryanodine Receptor Interaction with FKBP12 is Modulated by the RyR N-Terminus Repeat Region

Abstract

The ryanodine receptor (RyR) is an ion channel involved in the release of Ca2+ from intracellular stores (sarcoplasmic reticulum) in muscle cells. RyR plays a pivotal role in excitation-contraction coupling and is modulated by several accessory proteins in vivo, including the immunophilin FK506 binding protein 12 (FKBP12) which, our previous studies demonstrate, binds to RyR with extremely high affinity. The FKBP12 binding site within the RyR sequence has not been definitively identified and a number of candidate regions have been proposed. A central domain phosphorylation region of RyR (CDR) has been crystallised (Sharma, P. et al, FEBS (2013)288, 903-914; Yuchi, Z. et al., Structure (2012) 20, 1210-1211). This domain has considerable sequence and structural homology to its repeat region at the N-terminus (NTR) and a pseudo atomic model of the two repeat regions, docked onto a topology model of RyR, positions NTR adjacent to the previously proposed FKBP12 binding domain and suggests a potential interaction (Zhu, L. et al, JBC (2013) 288, 903-914). The RyR1 interaction with FKBP12 was tested using [3H]ryanodine binding to measure the Po of the channel of solubilised pig RyR preparations and microsomal preparations. A dose response to FKBP12 reduced activity by ∼ 40% at > 1µM. This inhibition was significantly reversed by the inclusion of 10µM recombinant NTR in the assay. NTR did not affect [3H]ryanodine binding in the absence of FKBP12. CDR recombinant protein was also tested, but this did not reverse FKBP12 inhibition. The results suggest that the NTR may play a role in the RyR interaction with FKBP12.