Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA051

Poster Communications

Single ryanodine receptor channels from the skeletal muscle of TRIC-A knockout mice show reduced activation by Mg2+ATP

K. Witschas1, S. El-Ajouz1, E. Venturi1, M. Beech1, F. O'Brien1, D. Eberhardt1, T. Iida2, M. Nishi2, H. Takeshima2, R. Sitsapesan1

1. Department of Pharmacology, University of Oxford, Oxford, United Kingdom. 2. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.

TRIC-A and TRIC-B are two related sarcoplasmic reticulum (SR) proteins that are thought to behave as monovalent cation channels (1, 2). The skeletal muscle from TRIC-A knockout mice is characterised by swollen, Ca2+ overloaded SR and impaired SR Ca2+ release (3). Since TRIC-A is the predominant TRIC subtype in skeletal muscle and is abundantly expressed, deletion of TRIC-A may affect ryanodine receptor (RyR1) function in ways additional to its ability to pass monovalent cation current across the SR. We therefore incorporated skeletal muscle SR vesicles into planar lipid bilayers under voltage-clamp conditions to compare the single-channel function of RyR1 derived from WT or TRIC-A KO skeletal muscle and used [3H]ryanodine binding assays to study populations of RyR1 channels as previously described (4). Mean values ± SEM were compared by Student's t test. With 10 µM cytosolic Ca2+ as the sole activator and Ca2+ as the permeant ion, we found no difference in RyR1 open probability (Po) from WT (0.002±0.001, n=36) and TRIC-A KO (0.002±0.003, n=14) mice, indicating that sensitivity to cytosolic Ca2+ is not altered. This was confirmed by comparing [3H]ryanodine binding to SR from WT and KO mice in the presence or absence of caffeine. However, single-channel recordings in the presence of 1 mM free Mg2+, showed that 3 mM ATP was significantly less effective at activating RyR1 from TRIC-A KO (Po=0.027±0.011, n=11) than from WT (Po=0.127±0.036, n=18, p<0.05) mice. The effects of Mg2+ATP were completely reversible for both groups of channel indicating that the increase in Po was not due to phosphorylation by an endogenous kinase that incorporated into the bilayer together with RyR1 but was induced by reversible ATP binding to the adenine nucleotide binding sites on RyR1. Adenine, a ligand that activates RyR1 via these nucleotide binding sites but which does not induce phosphorylation, stimulated [3H]ryanodine binding to the SR of both groups to a similar extent in the absence of Mg2+. However, Mg2+ inhibition of [3H]ryanodine binding to the SR from TRIC-A KO mice was significantly greater than that to the SR from WT mice. For example, at 10 µM Ca2+, 500 µM Mg2+ reduced [3H]ryanodine binding to 19.83±2.15% (n=6) of control values in TRIC-A KO SR compared to 29.30±3.44% (n=6, p<0.05) in WT SR. Our results indicate that TRIC-A can modulate RyR1 gating by affecting the ability of the channel to open in response to activating ligands in a manner that is not related to flux of monovalent current through SR K+ channels. The underlying mechanism appears to be due to altered sensitivity of RyR1 to Mg2+ inhibition. These results may, at least in part, explain why SR Ca2+-release is impaired in TRIC-A KO mice and demonstrate that TRIC-A may influence RyR1 function by multiple mechanisms. Funded by the BHF and JSPS.

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