Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC270
Description of the gating behaviour of purified human cardiac ryanodine receptor (hRyR2) by kinetic modelling and burst analysis under minimal conditions.
S. Mukherjee1, N. L. Thomas1, C. E. Maxwell1, A. J. Williams1
1. Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff, United Kingdom.
Rhythmic contraction of cardiac myocytes is maintained by precisely controlled Ca2+ efflux from intracellular stores mediated by the cardiac ryanodine receptor (RyR2)1. Mutations in RyR2 can cause channel instability leading to perturbed Ca2+ release that can trigger arrhythmias2. RyR2-dependent ventricular tachyarrhythmia is an important cause of sudden cardiac death, the mechanistic basis of which remains unclear. Most investigations of RyR2 single channel function have focussed on the secondary effects of mutation on single channel open probability (Po) via its modulation by regulatory proteins and cellular processes without emphasis on mutation-dependent effects on the gating behaviour of the channel per se. In this investigation we provide key novel mechanistic insights into the physical reality of RyR2 gating revealed by new experimental and analytical approaches. We have examined in detail the single channel gating kinetics of the purified hRyR2 when activated by cytosolic Ca2+ ([Ca2+]cyt) in a stringently regulated environment where the modulatory influence of factors external to the channel were minimised. Ca2+ activation of single purified recombinant hRyR2 channels was monitored in planar lipid bilayers3,4 where the luminal Ca2+ was buffered at 50 nM while the [Ca2+]cyt was stringently controlled using a cocktail of buffers to achieve an activating free [Ca2+]cyt range of nominally zero-500 μM. Single channel data were accurately analysed using hidden markov model (HMM) based algorithms in the QuB suite of analysis programs (www.qub.buffalo.edu). The resultant sigmoidal dose-response curve gave an EC50 of 1.45±0.44 μM (n=10) with Po saturating at ~10 μM Ca2+. The gating model generated describes the kinetic behaviour using a minimum of four open and three closed states and incorporates constitutive (unliganded) gating activity (Po: 1.09±0.5x10-5, open time: 0.17±0.06 ms; n=10) and reveals ligand-independent fast flicker closings transitions (τ: 0.38±0.03 ms, n=4) suggestive of gating events similar to C-type inactivation in K+ channels5. The kinetics also suggest an inhibitory role of [Ca2+]cyt beyond 1 μM where the backward ligand-dependent transitions were perturbed. Simulation using the putative gating models generated data similar to the actual and served to further validate the model. Novel detailed burst analysis of hRyR2 elucidates its ligand-bound gating kinetics where burst length increases and interburst interval decreases with increasing [Ca2+]cyt. This proposed model will serve as a benchmark against which the effects of disease causing mutations in hRyR2 can be studied, as well as the influence of physiological modulators and potentially therapeutic compounds capable of stabilising mutant RyR2 channel function.
Where applicable, experiments conform with Society ethical requirements