Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP₃R1) by cytosolic calcium (Ca²⁺) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP₃R1 regulation by Ca²⁺ are poorly understood. Using DT40 cell expression system and Ca²⁺ imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP₃R1 modulation by Ca²⁺. By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP₃R1 Ca²⁺-sensor region (E1932-R2270) binds Ca²⁺ with 0.16μM affinity. We further established that E2100D and E2100Q mutations decrease Ca²⁺-binding affinity of the putative InsP₃R1 Ca²⁺-sensor region to 1 μM. In planar lipid bilayer experiments with recombinant InsP₃R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP₃R1 bell-shaped Ca²⁺ dependence from 0.2 μM to 1.5 μM Ca²⁺. In agreement with the biochemical data, we found that the apparent affinities of Ca²⁺ activating and inhibitory sites of the InsP₃R1 were 0.2 μM for the wild-type channels and 1-2 μM Ca²⁺ for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP₃R1 Ca²⁺ sensor.