Functional determination of calcium-binding sites required for the activation of inositol 1,4,5-trisphosphate receptors

Abstract

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca²⁺) signals in response to various external cues. Notably, IP₃R channel activity is determined by several obligatory factors, including IP₃, Ca²⁺, and ATP. The critical basic amino acid residues in the N-terminal IP₃-binding core (IBC) region that facilitate IP₃ binding are well characterized. In contrast, the residues conferring regulation by Ca²⁺ have yet to be ascertained. Using comparative structural analysis of Ca²⁺-binding sites identified in two main families of intracellular Ca²⁺-release channels, ryanodine receptors (RyRs) and IP₃Rs, we identified putative acidic residues coordinating Ca²⁺ in the cytosolic calcium sensor region in IP₃Rs. We determined the consequences of substituting putative Ca²⁺ binding, acidic residues in IP₃R family members. We show that the agonist-induced Ca²⁺ release, single-channel open probability (P₀), and Ca²⁺ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues is neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca²⁺-binding pocket shifted the Ca²⁺ required to activate IP₃R to higher concentrations, indicating that these residues likely are a component of the Ca²⁺ activation site in IP₃R. Taken together, our findings indicate that Ca²⁺ binding to a well-conserved activation site is a common underlying mechanism resulting in increased channel activity shared by IP₃Rs and RyRs.

Keywords: calcium signaling; inositol 1,4,5 triphosphate receptor; ion channel regulation.