The high-affinity calcium - Calmodulin-binding site does not play a role in the modulation of type 1 inositol 1,4,5-trisphosphate receptor function by calcium and calmodulin

Elena Nosyreva, Tomoya Miyakawa, Zhengnan Wang, Lyuba Glouchankova, Akiko Mizushima, Masamitsu Iino, Ilya Bezprozvanny

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

Modulation of the inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R) by cytosolic calcium (Ca2+) plays an essential role in Ca2+ signalling, but structural determinants and mechanisms responsible for the InsP3R regulation by Ca2+ are poorly understood. In the present study, we expressed rat InsP3R type 1 (InsP3R1) in Spodoptera frugiperda cells using a baculovirus-expression system and reconstituted the recombinant InsP3R1 into planar lipid bilayers for functional analysis. We observed only minor effects of 0.5 mM of calmodulin (CAM) antagonist W-7 on the Ca2+ dependence of InsP3R1. Based on a previous analysis of mouse InsP3R1 [Yamada, Miyawaki, Saito, Nakajima, Yamamoto-Hino, Ryo, Furuichi and Mikoshiba (1995) Biochem J. 308, 83-88], we generated the Trp1577 → Ala (W1577A) mutant of rat InsP3R1 which lacks the high-affinity Ca2+-CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Ca2+ dependence similar to the wild-type InsP3R1 in planar lipid bilayers. Activation of B cell receptors resulted in identical Ca2+ signals in intact DT40 cells lacking the endogenous InsP3R and transfected with the wild-type InsP3R1 or the W1577A mutant cDNA subcloned into a mammalian expression vector. In the planar lipid bilayer experiments, we showed that both wild-type InsP3R1 and W1577A mutant were equally sensitive to inhibition by exogenous CaM. From these results, we concluded that the interaction of CaM with the high-affinity Ca2+-CaM-binding site in the coupling domain of the InsP3R1 does not play a direct role in biphasic modulation of InsP3R1 by cytosolic Ca2+ or in InsP3R1 inhibition by CaM.

Original languageEnglish (US)
Pages (from-to)659-667
Number of pages9
JournalBiochemical Journal
Volume365
Issue number3
DOIs
StatePublished - Aug 1 2002

Keywords

  • Baculovirus
  • Calcium signalling
  • DT40 cells
  • Planar lipid bilayer
  • Sf9 cells
  • Single-channel recordings
  • Structure function

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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