The high-affinity calcium[bond]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 (Ca(2+)) plays an essential role in Ca(2+) signalling, but structural determinants and mechanisms responsible for the InsP(3)R regulation by Ca(2+) 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 Ca(2+) 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 Ca(2+)[bond]CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Ca(2+) dependence similar to the wild-type InsP(3)R1 in planar lipid bilayers. Activation of B cell receptors resulted in identical Ca(2+) 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(3)R1 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 Ca(2+)[bond]CaM-binding site in the coupling domain of the InsP(3)R1 does not play a direct role in biphasic modulation of InsP(3)R1 by cytosolic Ca(2+) or in InsP(3)R1 inhibition by CaM.
Nosyreva
Elena
E
Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, U.S.A.
Miyakawa
Tomoya
T
Wang
Zhengnan
Z
Glouchankova
Lyuba
L
Mizushima
Akiko
A
Iino
Masamitsu
M
Bezprozvanny
Ilya
I
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008051:Lipid Bilayers; D016297:Mutagenesis, Site-Directed; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013449:Sulfonamides / Q000494:pharmacology
D015220:Calcium Channels; D002147:Calmodulin; D004791:Enzyme Inhibitors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008051:Lipid Bilayers; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013449:Sulfonamides; C017967:W 7; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium