Bcl-xL Regulation of InsP3 Receptor Gating Mediated by Dual Ca2+ Release Channel BH3 Domains

Interaction of anti-apoptotic Bcl-xL with inositol trisphosphate receptor (InsP3R) Ca2+ release channels sensitize them to InsP3, enhancing low-level constitutive Ca2+ signaling that affords apoptosis resistance (White et al. Nat Cell Biol 7(2005); Li et al. PNAS 104(2007)). Here, we have identified two binding domains in the channel C-terminus that are both required for channel activation: the first is located at the bottom of TM6 immediately distal to the gate, and the second is near the extreme C-terminus. Each site binds to anti-apoptotic Bcl-2, Bcl-xL and Mcl-1 with similar affinities, but not to pro-apoptotic Bid or Bax. Bcl-xL binding to a construct containing both sites had apparent affinity several times higher than for either individual site or for Bax. Bcl-xL interaction with Bcl-2 proteins is mediated by pro-apoptotic protein BH3 domains. Several features suggest that each binding site in the InsP3R is similar to a BH3 domain. Sequence and secondary structural features are reminiscent of BH3 domains; mutations of key residues known to disrupt BH3 domain interactions disrupted Bcl-XL binding to either channel site; mutations in Bcl-xL that disrupt binding to BH3 domains inhibited binding to the channel sites; ABT-737 that binds in the Bcl-XL BH3 binding pocket inhibited binding to each channel site. In single-channel recordings, Bcl-xL activation of gating was abolished by mutations of either channel BH3-like domain or of the Bcl-xL BH3 binding pocket, or by ABT-737. These results suggest that dimeric Bcl-xL cross-links TM6 and the distal C-terminus through interactions involving channel BH3 domains. This interaction allosterically enhances the channel sensitivity to InsP3 by enabling the channel to open more easily.