The human cardiac ryanodine receptor gating behaviour: a study of mechanisms

Rhythmic contraction of cardiac myocytes is maintained by precisely controlled Ca2+ efflux from intracellular stores mediated by the cardiac ryanodine receptor (RyR2). Mutations in RyR2 result in perturbed Ca2+ release that can trigger arrhythmias. RyR2- dependent ventricular tachyarrhythmia is an important cause of sudden cardiac death, the mechanistic basis of which remains unclear. RyR2 dysfunction has also been implicated in other cardiovascular disorders such as heart failure and cardiomyopathy, thereby becoming an important target for putative drugs. The massive size of RyR2 (~2.2 MDa) along with its intracellular location poses considerable challenges to studies aimed at understanding the mechanisms underlying channel dysfunction. Single channel studies of reconstituted RyR2 in artificial lipid bilayers have provided important insights into channel behaviour in response to various physiological ligands, toxins, drugs and biochemical modifications. However, the precise mechanisms by which RyR2 is activated by its primary physiological trigger, cytosolic Ca2+, and the structural determinants of channel gating are yet unknown. In this study, I aim to understand the actual physical reality of RyR2 gating behaviour using novel experimental approaches and analytical procedures. I have examined in detail, single channel kinetics of wild type purified recombinant human RyR2 (hRyR2) when activated by cytosolic Ca2+ in a precisely regulated minimal environment where the modulatory influence of factors external to the channel were minimised. This mathematical modelling of hRyR2 single channel behaviour will serve as a future experimental platform upon which the effects of disease causing mutations can be studied, as well as the influence of physiological modulators and potentially therapeutic compounds capable of stabilising mutant channel function. Single channel studies of hRyR2 when modified by its archetypal ligand ryanodine in the absence of Ca2+ have uncovered an unusual voltage sensitive gating behaviour in this ligand-gated channel, providing further insights into the mechanisms underlying channel modification