Ryanodine receptors are targeted by anti-apoptotic Bcl-X(L) involving its BH4 domain and Lys87 from its BH3 domain

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca(2+)-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), while Bcl-X(L), likely independently of its BH4 domain, sensitizes IP(3)Rs. It remains elusive whether Bcl-X(L) can also target and modulate RyRs. Here, Bcl-X(L) co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-X(L) bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-X(L) to bind to RyRs, loading the BH4-Bcl-X(L) peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca(2+) release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-X(L) indicated that Lys87 of the BH3 domain of Bcl-X(L) could be important for interacting with RyRs. In contrast to Bcl-X(L), the Bcl-X(L)(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca(2+) release. These data suggest that Bcl-X(L) binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction.