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

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