Normal rhythmic contraction of the myocardium depends on the controlled release of Ca2+ from intracellular stores via cardiac ryanodine receptors (RyR2). Mutations in RyR2 are linked with arrhythmia and give rise to catecholaminergic polymorphic ventricular tachycardia (CPVT)1. A consensus view on the mechanisms of RyR2 dysfunction in CPVT proposes that mutant channels exhibit gain-of-function activity due, at least in part, to abnormal interactions with, or enhanced sensitivities to modulatory ligands2. We have previously described the gating of wild type (WT) human RyR2 channel in response to cytosolic Ca2+ 3, resulting in a gating scheme, which includes a number of gating phenomena never before described for RyR2 and serves as a platform for modelling the effects of disease-linked mutations on the channel. In the present study, we used identical minimal experimental conditions (i.e. with [K+] at 210mM as the permeant ion, using purified recombinant human channels devoid of regulatory co-proteins) in order to characterize molecular defects in exemplar CPVT-linked mutant channels. Data are given as mean±SEM and compared using t-tests or ANOVA where appropriate. In contrast to WT channels and a central domain mutation (S2246L), the N4104K mutation exhibited a higher frequency of gating events in the absence of Ca2+. These data suggest that the N4104K mutation introduces intrinsic instability in the channel structure. This concept was explored further using Hidden Markov Modelling of N4104K gating data using QuB4 which revealed that this propensity for unliganded gating was likely due to an increased susceptibility of N4104K channels to enter a conformational state that favours subsequent opening. This was quantified as a decrease in the ratio of isomerisation between responsive and non-responsive closed states found in our model, Eiso: WT = 0.42±0.14 vs N4104K = 0.07±0.06 (n= 2-6). Confocal imaging of spontaneous Ca2+ release events in fluo-3 loaded HEK293 cells expressing WT or mutant RyR2s demonstrated that both CPVT-linked mutations resulted in a gain-of-function with both S2246L and N4104K showing an increased duration of Ca2+ release event compared to WT (WT 7.97±0.25 s, vs SL 9.01±0.41 s, NK 10.52±0.61 s, p<0.05, n = 17-46). However, cells expressing these CPVT mutants exhibited markedly different inter-transient durations (decreased for NK 3.82±0.76 s, increased for SL 8.47±0.73 s vs WT 6.46±0.50 s, p<0.05, n = 17-46) compared to WT. The data suggest that a fundamental conformational defect gives rise to unliganded gating in N4104K channels which leads to an increased propensity for Ca2+ release in cellular systems. These data point to the likelihood that the generic assignment of CPVT-linked RyR2 mutations as ‘gain-of-function’ masks different mutant modes of RyR2 channel dysfunction.