| Ryanodine receptors (RyR) are large intracellular calcium release channels, which regulate calcium homeostasis, and play a vital role in excitation-contraction coupling (ECC). RyR channel gating is controlled in part by inter-domain interactions between the N-terminal and central mutation-rich region. Adhesion of these domains to one anotheris thought to stabilize the RyR closed state whereas and separation of theses domains is thought to stabilize the RyR open state. To gain insight into the biophysical mechanism underlining inter-domain interactions in RyR1, the effects that CHAPS and a short domain peptide, Dp4, (which are thought to disrupt inter-domain interactions, and activate RyR) have on purified RyR1 were characterized by single-particle cryo-electron microscopy (cryo-EM), and by a [3H]ryanodine binding assay. Results of these experiments showed that both CHAPS and Dp4 activated RyR in membrane fraction of the sarco(endo)plasmic reticulum (SR/ER) but had no effect on purified RyR1, and did not appear to alter the ultrastructure of RyR1.;ECC is mediated by a large and dynamic macromolecular complex often referred to as the ECC machinery; the core of this complex consists of RyR and dihydropyridine receptor (DHPR), along with several other smaller regulatory proteins (e.g. FK 506 binding protein, calsequestrin, calmodulin). I have developed a protocol for cross-linking various proteins to RyR1, which could be used in conjunction with single-particle cryo-EM to characterize the structure of labile RyR complexes consisting of RyR and one or more other components of the ECC machinery. As a proof of principle, the His-FKBP12:RyR1 complex was used as a model system to assess the cross-linking protocol. Glutaraldehyde effectively cross-linked FKBP12 to RyR1 while preserving the overall morphology of RyR1. Chemical cross-linking did induce minor changes to the cytoplasmic and transmembrane assemblies. Furthermore, glutaraldehyde cross-linking enhanced [3 H]ryanodine binding to purified RyR1. Based on these findings I postulate that glutaraldehyde chemical cross-linking locks RyR1 in an open-like conformational state. |
