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 (InsP(3)) receptors (InsP(3)R) by cytosolic calcium (Ca2+) plays an essential role in Call signalling, but structural determinants and mechanisms responsible for the InsP(3)R regulation by Call are poorly understood. In the present study, we expressed rat InsP(3)R type 1 (InsP(3)R1) in Spodoptera frugiperda cells using a baculovirus-expression system and reconstituted the recombinant InsP(3)R1 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 InsP(3)R1. Based on a previous analysis of mouse InsP(3)R1 [Yamada, Miyawaki, Saito, Nakajima, Yamamoto-Hino, Ryo, Furuichi and Mikoshiba (1995) Biochem J. 308, 83-88], we generated the Trp(1577) --> Ala (W1577A) mutant of rat InsP(3)R1 which lacks the high-affinity Ca2+-CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Call dependence similar to the wild-type InsP,RI in planar lipid bilayers. Activation of B cell receptors resulted in identical Call signals in intact DT40 cells lacking the endogenous InsP(3)R and transfected with the wild-type InsP(3)R1 or the W1577A mutant cDNA subcloned into a mammalian expression vector. In the planar lipid bilayer experiments, we showed that both wild-type InsP,RI 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 InsP,RI does not play a direct role in biphasic modulation of InsP(3)R1 by cytosolic Ca2+ or in InsP(3)R1 inhibition by CaM.