Precisely controlled Ca2+ release from the sarcoplasmic reticulum in the cardiomyocyte, mediated by the cardiac ryanodine receptor Ca2+ release channel (RyR2), is essential for rhythmic contraction of the heart1.In spite of the paucity of detailed structural information for the RyR2 pore and the gating entities it contains, functional studies have indicated the possibility of two gates2. The Ca2+ (ligand) controlled gate at the inner helix bundle crossover (IHBx) region of the RyR2 pore was found to be mechanistically distinct from that of the gating at the selectivity filter (SF) region that is Ca2+ independent2.As ion channel blockers have previously been shown to be effective antiarrhythmic agents3, it is envisaged that a similar mechanism of drug action could be employed in the case of RyR2 dependent arrhythmia. This study looks at the effect of RyR2 channel block by large quaternary ammonium cations as prototypical blockers to unravel any functional consequences of block on channel gating.Single channel studies were conducted after reconstituting purified, recombinant, mouse RyR2 into artificial planar lipid bilayers. The blockers tetrabutyl- and pentyl ammonium (TBA & TPeA) were added to the cytosolic side of channels that were activated by Ca2+ and EMD 41000 and blocker kinetics measured with K+ as the permeant ion4.The blocking events (to subconductance levels) were only observed at positive holding potentials (cytosolic to luminal ionic flux). When single channel open probabilities (Po) measured at negative holding potentials are plotted against probabilities of block at corresponding positive potentials, the intercept of the extrapolated graph suggests that the blocker could also access a closed conformation of RyR2. This demonstrates that although the opening of the ligand-controlled gate formed by IHBx enables blockers to access the pore, the channel can still be blocked from the closed conformation when closings are due to gating at the SF region. Analyses of state transitions between various closed, blocked and open states were used to determine the effects of pore block on channel gating. The frequency of flicker closing events (τC=0.40±0.03 ms; Area = 39±6 %, n = 5) is altered in the presence of blocker (e.g. for TPeA, τCb=0.44±0.13 ms; Area = 66±4.7 %, n = 4), with the channel visiting only one predominant open state (τO=4.56±0.58 ms, n=4) as opposed to two. This increase in the frequency of closing events is likely due to the blockers’ ability to reduce the availability of permeant ions in the pore, previously shown to stabilise the SF, preventing its collapse in other ion channels5. This study demonstrates that RyR2 blockers could inhibit channel activity due to their effects on gating in addition to reducing single channel current by physical block of the pore. Values are means ± SEM, data compared by t-test.