CaBP1, a neuronal Ca ^{2 +} sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions

Abstract

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca 2+ channels, including inositol 1,4,5-trisphosphate receptors (InsP 3 Rs). CaBP1 alone does not affect InsP 3 R activity, but it inhibits InsP 3 -evoked Ca 2+ release by slowing the rate of InsP 3 R opening. The inhibition is enhanced by Ca 2+ binding to both the InsP 3 R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP 3 R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca 2+ binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the β-domain of the InsP 3 -binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP 3 R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP 3 R pore. InsP 3 activates InsP 3 Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a "hot-spot" loop in the suppressor domain (residues 1-223) and the InsP 3 -binding core β-domain. Targeted cross-linking of residues that contribute to this interface show that InsP 3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP 3 R activity by restricting the intersubunit movements that initiate gating.

Structure of IP3R channel: high-resolution insights from cryo-EM

“…During the past decade, structural information on the IP 3 R protein has been obtained by three major structural techniques: X-ray crystallography [28–31], NMR spectroscopy [32,33] and single-particle cryo-EM [19,34–40]. However, given the exceptionally large size of the entire IP 3 R channel (~1.2 MDa), only single particle cryo-EM was able to tackle the structure of the full-length tetrameric channel assembly.…”

Intracellular calcium channels: Inositol-1,4,5-trisphosphate receptors

“…In aggregate with data demonstrating that Ca 2+ -CaBP1 inhibits InsP 3 -evoked Ca 2+ release (Haynes et al, 2004; Kasri et al, 2004b; Li et al, 2013) a novel regulatory molecular mechanism has been proposed where CaBP1 clamps the intersubunit interactions and thereby inhibits channel opening (Fig. 4C) (Li et al, 2013). …”

“…Importantly, Li et al produced covalently linked tetrameric InsP 3 R1-NT through site-specific cysteine insertions in the intersubunit interface that was modeled in the docking studies and demonstrated that InsP 3 inhibits cross-linked tetrameric InsP 3 R1-NT in a concentration-dependent manner, suggesting that InsP 3 binding closes the clam-like IBC, disrupting these intersubunit interactions and allowing the channel to open (Fig. 4C) (Li et al, 2013). Taken together, these observations indicate that the InsP 3 R1-NT plays a critical role in the allosteric modulation of ion channel conductance through a modification of quaternary arrangement.…”

“…4A) (Li et al, 2013). The structure showed that exposed hydrophobic residues in Ca 2+ -bound CaBP1 ( i.e.…”

“…4B revealed that the four molecules of CaBP1 form a ring-like structure around the central cytosolic vestibule. Further, chemical cross-linking experiments showed that CaBP1 enhances the production of cross-linked tetrameric InsP 3 R1-NT (Li et al, 2013). In aggregate with data demonstrating that Ca 2+ -CaBP1 inhibits InsP 3 -evoked Ca 2+ release (Haynes et al, 2004; Kasri et al, 2004b; Li et al, 2013) a novel regulatory molecular mechanism has been proposed where CaBP1 clamps the intersubunit interactions and thereby inhibits channel opening (Fig.…”