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

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.