ABSTRACT
The anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein not only counteracts apoptosis at the mitochondria by scaffolding pro-apoptotic Bcl-2-family members, but also acts at the endoplasmic reticulum, thereby controlling intracellular Ca2+ dynamics. Bcl-2 inhibits Ca2+ release by targeting the inositol 1,4,5-trisphosphate receptor (IP3R). Sequence analysis has revealed that the Bcl-2-binding site on the IP3R displays strong similarity with a conserved sequence present in all three ryanodine receptor (RyR) isoforms. We now report that Bcl-2 co-immunoprecipitated with RyRs in ectopic expression systems and in native rat hippocampi, indicating that endogenous RyR–Bcl-2 complexes exist. Purified RyR domains containing the putative Bcl-2-binding site bound full-length Bcl-2 in pulldown experiments and interacted with the BH4 domain of Bcl-2 in surface plasmon resonance (SPR) experiments, suggesting a direct interaction. Exogenous expression of full-length Bcl-2 or electroporation loading of the BH4 domain of Bcl-2 dampened RyR-mediated Ca2+ release in HEK293 cell models. Finally, introducing the BH4-domain peptide into hippocampal neurons through a patch pipette decreased RyR-mediated Ca2+ release. In conclusion, this study identifies Bcl-2 as a new inhibitor of RyR-based intracellular Ca2+-release channels.
Author contributions
The study was conceived and originally designed by T.V., H.D.S., J.B.P. and G.B., with subsequent input by N.N.K for the use of hippocampal neurons. T.V. and E.D. performed the experiments. J.M. and V.S. contributed essential reagents and materials. T.V., E.D., L.M., L.L., H.D.S., N.N.K., J.B.P. and G.B. analyzed, interpreted and/or discussed the data. T.V. and G.B. drafted the manuscript. All authors critically revised the manuscript and approved the final article.
Funding
This work was supported by the Research Foundation-Flanders (FWO) [grant numbers G.0571.12 to G.B., H.D.S., J.B.P. and L.L., and G.0134.09N to L.L.]; by the Research Council of the KU Leuven (OT START) [grant number STRT1/10/044 to G.B.]; by the Interuniversity Attraction Poles Program (Belgian Science Policy) [grant numbers P7/13 to J.B.P., G.B. and L.M., and P7/10 to L.L.]; and by the Donders Center for Neuroscience fellowship award of the Radboud University Nijmegen Medical Center; and the FP7-Marie Curie International Reintegration Grant [grant number 277091] to N.N.K. T.V. was supported for his stay at the Radboud University by FWO travel grant [grant number V42613N].
