The cardiac ryanodine receptor (RyR2) P2328S mutation is a cause of both catecholaminergic polymorphic ventricular tachycardia (CPVT) [1], leading to fatal ventricular arrhythmias, and atrial arrhythmias including atrial fibrillation. In vivo, RyR2 can exist in various phosphorylation states and in association with regulatory proteins such as FKBP12/12.6 thought to affect channel stability [2,3]. Wild-type (WT) and homozygous RyR2-P2328S inbred 129/Sv mice, age matched across 3-7 months were killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act 1986; hearts were rapidly excised and snap frozen in liquid N2. Homotetrameric RyR2-containing sarcoplasmic reticular (SR) vesicle preparations (each 5-7 hearts) were incorporated into lipid bilayers for single channel recordings and used for co-immunoprecipitation (Co-IP) and western blot (WB). Single channel recordings assessed open probability (Po), mean open time, mean closed time and event frequency at a range of cytoplasmic [Ca2+] ([Ca2+]cyto); 0.1 µM – 1 mM. Recordings with multiple channels were only used for I’F (mean current normalised to maximum current – Po equivalent) determination. Anti-RyR2 Co-IPs were performed and samples run on SDS-PAGE, transferred to PVDF and probed with antibodies for RyR2, FKBP12/12.6 as well as the S2808 and S2814 phosphorylated forms of RyR2. Data are means ± SEM compared by student’s t-test (channel data) or ANOVA with Tukey post hoc (Western blots). Concentration dependences of Po were fit to a Hill equation. Activity in WT and P2328S RyR2 channels (Fig 1) was similar at 1 mM [Ca2+]cyto (Po at -40 mV; 0.55±0.08 vs 0.56±0.07, respectively), but P2328S was significantly more active than WT at 1 µM [Ca2+]cyto (0.24±0.08 vs 0.028±0.07, respectively). This was associated with a >10-fold shift in the AC50 for Ca2+-activation from ~3.5 µM Ca2+ in WT RyR2 to ~320 nM in P2328S channels and an unexpected >1000-fold shift in IC50 for inactivation from ~50 mM in WT channels to ≤7μM in P2328S channels, within systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+-activation was not associated with changes in S2808 or S2814 phosphorylation, or FKBP12/12.6 bound to the channels. These data demonstrate that in vivo derived P2328S-RyR2 channels are more sensitive to cytoplasmic Ca2+ concentrations within the lower diastolic range (0.1-1 µM). This was in the absence of adrenergic challenge and without altered FKBP12 binding or RyR2 S2808/S2814 hyperphosphorylation. This suggests an increased predisposition to CPVT and an alternative mechanism for enhanced channel activity not dependent on hyperphosphorylation or loss of FKBP12/12.6-mediated stabilisation of RyR2 channels.