Human cardiac ryanodine receptors (hRyR2) play an indispensible role in excitation-contraction coupling, allowing Ca2+ to pass from the sarcoplasmic reticulum (SR), into the cytoplasm of cardiomyocytes. These Ca2+ release channels form quaternary complexes with luminal accessory proteins calsequestrin (CSQ2), junctin (JUN) and triadin (TRD1), which are thought to regulate the response of hRyR2 to changes in luminal Ca2+ within the SR1. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an arrhythmogenic disorder, caused by mutations in hRyR22 and CSQ23 genes and defective channel sensing of cytosolic and luminal Ca2+ plays an important role in disease pathogenesis4,5. Given the proposed role of CSQ2 and JUN in modulating the response of hRyR2 channels to activating Ca2+, altered association or dysfunctional regulation by these accessory proteins could be a contributing factor in the aberrant Ca2+ release identified in CPVT-linked mutant hRyR2. In this investigation we examine the interaction and response of wild-type (WT) and CPVT-linked mutant (A4556T and N4104K) eGFP-hRyR2 channels to CSQ2 and JUN co-expression in HEK293 cells. Luminal Ca2+ effects were monitored by examination of spontaneous Ca2+ release (SCR) events4 in fluo-3 loaded cells using confocal microscopy. Interaction with hRyR2 was assessed using pixel co-localisation of the co-expressed eGFP-hRyR2 with Alexa-594 labelled CSQ/JUN in fixed cells. All values are given as mean±SEM and analysed using one-way analysis of variance and a tukey-kramer post-hoc test (GraphPad Prism). Demonstrating functional heterogeneity between hRyR2 mutations, A4556T-hRyR2 displayed a similar pattern of SCR to WT, whilst N4104K-hRyR2 displayed a remarkably altered profile characterised by an increased frequency of events (WT 2.1±0.15 vs. NK 8.1±1.5 events/min, p<0.001 n=14) and decreased amplitude (ΔF/F0 WT 1.59±0.16 vs NK 0.79±0.08, p<0.05 n=12). Co-expression with CSQ2 (either in the presence or absence of JUN) had inhibitory effects on WT and N4104K-hRyR2, increasing inter-event duration (WT 36±3.69 vs NK 7.31±0.96 secs, p<0.001 n=13), thereby decreasing SCR event frequency (WT 1.34±0.13 vs NK 3.10±0.29 events/min, p<0.001 n=14), yet this inhibitory effect was not observed with A4556T-hRyR2 (AT+CSQ2 2.43±0.28 vs AT only 2.37±0.29 events/min n=14), suggesting that this CPVT mutant is unable to be regulated by this accessory protein. In agreement with this, immunofluorescent co-incident pixel counting suggested reduced CSQ2 and JUN association with A4556T-hRyR2 compared to WT (for example, WT 93%±1.46 vs AT 80%±3.0, p<0.05 n=12 (CSQ2 association in cells expressing hRyR2+CSQ2+JUN), whereas association with N4104K-hRyR2 was unchanged (85%±6.75). This suggests that A4556T affects hRyR2 regulation by CSQ2, possibly as a result of defective protein-protein interaction.