Several members of the anti-apoptotic Bcl-2-protein family, including Bcl-2, Bcl-X-L and Mcl-1, directly bind and regulate the inositol 1,4,5-trisphosphate receptor (IP3R), one of the two main intracellular Ca2+-release channel types present in the endoplasmic reticulum. However, the molecular determinants underlying their binding to the IP3R remained a matter of debate. One interaction site for Bcl-2 was proposed in the central part of the modulatory domain [Y.P. Rong, AS. Aromolaran, G. Bultynck, F. Zhong, X. Li, K. McColl, S. Matsuyama, S. Herlitze, H.L. Roderick, M.D. Bootman, G.A. Mignery, J.B. Parys, H. De Smedt, C.W. Distelhorst, Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals, Mol. Cell 31 (2008) 255-265] and another site in the C-terminal domain of the IP3R encompassing the sixth transmembrane domain, to which Bcl-2, Bcl-X-L and Mcl-1 can bind [E.F. Eckenrode, J. Yang, G.V. Velmurugan, J.K. Foskett, C. White, Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca2+ signaling, J. Biol. Chem. 285 (2010) 13678-13684]. Here, we investigated and compared the binding of Bcl-2 and Bcl-X-L to both sites. Two different IP3R domains were used for the C-terminal site: one lacking and one containing the sixth transmembrane domain. Our results show that elements preceding the C-terminal cytosolic tail located at the sixth transmembrane domain of IP(3)R1 were critical for recruiting both Bcl-2 and Bcl-X-L to the C-terminal part of the IP3R. Furthermore, consistent with our previous observations, Bcl-X-L bound with higher efficiency to the C-terminal part of the IP3R and to a much lesser extent to the central, modulatory domain, while Bcl-2 targeted both sites with similar efficiencies. In conclusion, IP3R harbors two different binding sites for anti-apoptotic Bcl-2 proteins, one in the central, modulatory domain and one in the C-terminal domain near the Ca2+-channel pore. (C) 2012 Elsevier Inc. All rights reserved.