A mechanistic description of gating of the human cardiac ryanodine receptor in a regulated minimal environment

Cardiac muscle contraction, triggered by the action potential, is mediated by the release of Ca(2+) from the sarcoplasmic reticulum through ryanodine receptor (RyR)2 channels. In situ, RyR2 gating is modulated by numerous physiological and pharmacological agents, and altered RyR2 function underlies the occurrence of arrhythmias in both inherited and acquired diseases. To understand fully the mechanisms underpinning the regulation of RyR2 in the normal heart and how these systems are altered in pathological conditions, we must first gain a detailed knowledge of the fundamental processes of RyR2 gating. 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 human RyR2 when activated by cytosolic Ca(2+) in a stringently regulated environment where the modulatory influence of factors external to the channel were minimized. The resulting gating schemes are based on an accurate description of single-channel kinetics using hidden Markov model analysis and reveal several novel aspects of RyR2 gating behavior: (a) constitutive gating is observed as unliganded opening events; (b) binding of Ca(2+) to the channel stabilizes it in different open states; (c) RyR2 exists in two preopening closed conformations in equilibrium, one of which binds Ca(2+) more readily than the other; (d) the gating of RyR2 when bound to Ca(2+) can be described by a kinetic scheme incorporating bursts; and (e) analysis of flicker closing events within bursts reveals gating activity that is not influenced by ligand binding. The gating schemes generated in this investigation provide a framework for future studies in which the mechanisms of action of key physiological regulatory factors, disease-linked mutations, and potential therapeutic compounds can be described precisely.