Association between RyR and FKBP in experimental preparations: evidence for negative cooperativity

Abstract

Ryanodine receptors (RyR), a class of massive tetrameric intracellular ion channels, are an indispensable element of excitation-contraction coupling, governing the release of Ca2+ from the sarcoplasmic reticulum (SR). FK506-binding proteins (FKBP), a class of soluble proteins named for their affinity to the immunosuppressant drug FK506, have long been known to bind RyR endogenously, at a stoichiometry of one FKBP per RyR monomer. Much is unknown regarding the relationship between the two species, however: for example, it is not known whether FKBP-binding sites on the RyR are fully occupied in normal physiology, or whether instead there might be a dynamic aspect to the association between the two proteins. In this thesis, the relationship between the two most prominent isoforms of RyR, RyR1 and RyR2, and their endogenous ligands, FKBP12 and FKBP12.6, is examined via biochemical experiments. The effects of FKBP-dissociating treatments, saturation of RyR by exogenous FKBP, and the effects of the FKBP-RyR stabilising drug S107 are all examined in depth, with additional observations made on the dimerisation of GST-FKBP12 and FKBP12 and the effects of SR vesicle processing on the association between RyR and FKBP. These experiments give rise to some surprising results: FKBP-dissociating treatments have very different effects on RyR1 and RyR2, and while considerable FKBP is lost from RyR during the preparation of SR vesicles, it appears that RyR may not be endogenously saturated with FKBP. Finally, drawing on observations made during the biochemical experiments comprising this thesis, one particularly interesting model of a relationship between RyR and FKBP will be explored in greater depth: negative cooperation between the two species, wherein each molecule of FKBP binding to a RyR tetramer renders any subsequent binding less likely. This model is reconciled with the existing literature, and intriguing preliminary data is presented in support of negative cooperativity. Show more