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.
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