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Functional and Biochemical Analysis of the Type 1 Inositol (1,4,5)-Trisphosphate Receptor Calcium Sensor

Huiping Tu ^*, Elena Nosyreva ^*, Tomoya Miyakawa , Zhengnan Wang ^*, Akiko Mizushima , Masamitsu Iino  and Ilya Bezprozvanny ^*

^* Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas USA; and Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

Correspondence: Address reprint requests to Dr. Ilya Bezprozvanny, Dept. of Physiology, K4.112, UT Southwestern Medical Ctr. at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Tel.: 214-648-6737; Fax: 214-648-2974; E-mail: Ilya.Bezprozvanny{at}UTSouthwestern.edu.

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 0.16 µM 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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