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, Ilya Bezprozvanny

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)290-299
Number of pages10
JournalBiophysical Journal
Volume85
Issue number1
Publication statusPublished - Jul 1 2003

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ASJC Scopus subject areas

  • Biophysics

Cite this

Tu, H., Nosyreva, E., Miyakawa, T., Wang, Z., Mizushima, A., Iino, M., & Bezprozvanny, I. (2003). Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor. Biophysical Journal, 85(1), 290-299.