The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca(2+). A synthetic peptide corresponding to part of the central domain (DPc10:(2460)G-P(2495)) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca(2+) as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca(2+) and a large increase in the magnitude of luminal Ca(2+) activation. The increase in the luminal Ca(2+) response appeared reliant on the luminal-to-cytoplasmic Ca(2+) flux in the channel, indicating that luminal Ca(2+) was activating the RyR2 via its cytoplasmic Ca(2+) sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca(2+) sensing sites. The results were fitted by the luminal-triggered Ca(2+) feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca(2+)-activation mechanism.