Identifying the in vitro interactions between the skeletal muscle ryanodine receptor, triadin and calsequestrin
Creators: name: Wium, Elize
Publisher: name: The Australian National University
Year: 2014
Subjects: subject: Ryanodine Receptors
subject: Muscle contraction
subject: Muscle proteins

description: Excitation-contraction coupling (EC coupling) is a process linking depolarization of a muscle fibre membrane with eventual mechanical contraction of that fibre. Key in this process is calcium release into the muscle fibre cytoplasm from an intracellular store (sarcoplasmic reticulum, SR) via a ligand gated calcium channel, the ryanodine receptor (RyR). Numerous proteins engage in RyR regulation to ensure healthy calcium release during EC coupling. One such protein is triadin, which binds both the cytoplasmic and SR luminal domains of the RyR. Triadin has been shown to engage in direct RyR regulation in vitro, however, in vivo it has been associated with many roles not directly related to RyR regulation or EC coupling. As such, it has proven difficult to correlate triadin's protein-protein interactions to functional effects in vivo. In an attempt to better understand the nature of the luminal triadin-RyR association, regulatory interactions between the major rabbit skeletal muscle triadin isoform, Trisk 95, and the skeletal RyR isoform (RyR1) were investigated. Using a triadin peptide corresponding to residues 200 to 232 of rabbit Trisk 95, it was found that this region was sufficient to replicate specific RyR1 activation as mediated by full length Trisk 95 in vitro. Furthermore, using a set of mutant peptides, it was discovered that K218, K220 and K224 were minimally required to recapitulate full length Trisk 95 mediated RyR1 regulation. Peptides incorporating only one or two of these mutations retained RyR1 regulation, but highlighted a tendency for K220 to be more critical in maintaining RyR1 regulation than K218 and K224. This matched well with previous work showing three RyR1 residues contribute unequally to Trisk 95 binding. Consequently, it was hypothesised that the luminal association between Trisk 95 and RyR1 requires three charged pair interactions, with each contributing differently to the overall interaction. It is hoped that the identification of Trisk 95's RyR1 binding residues will enable specific mutational targeting of the luminal association between Trisk 95 and RyR1 in future studies, thereby identifying the function of this interaction in vivo. Interestingly, one of Trisk 95's other protein binding partners, calsequestrin (CSQ1), has been predicted to bind Trisk 95 at residues overlapping those here identified as critical for RyR1 regulation. Investigating whether the same Trisk 95 residues critical for RyR1 regulation are also critical for CSQ1 binding, it was found that a triadin peptide incorporating mutation of K218, K220 and K224 was still able to bind substantial amounts of CSQ1. This suggested that Trisk 95 binds RyR1 and CSQ1 via different residues. However, the RyR1 regulating and CSQ1 binding residues were in close proximity, leaving the possibility that CSQ1 or RyR1 may occlude the other's binding site on Trisk 95 when both are present. Consequently, a model is proposed to explain how an inability of Trisk 95 to simultaneously bind RyR1 and CSQ1 might result in dynamic RyR1 regulation. The model presents a new set of hypotheses which, upon testing, are expected to expand current knowledge regarding Trisk 95's role in EC coupling.