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
| ISSN: | 1470-8728 0264-6021 |
|---|---|
| DOI: | 10.1042/bj20011789 |
| Přístupová URL adresa: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f237f8887ba80f97585cebe734e2807d https://doi.org/10.1042/bj20011789 |
| Rights: | OPEN |
| Přírůstkové číslo: | edsair.doi.dedup.....f237f8887ba80f97585cebe734e2807d |
| Autor: | Elena Nosyreva, Masamitsu Iino, Tomoya Miyakawa, Akiko Mizushima, Ilya Bezprozvanny, Lyuba Glouchankova, Zhengnan Wang |
| Rok vydání: | 2002 |
| Předmět: |
endocrine system
Calmodulin Lipid Bilayers Mutant Receptors Cytoplasmic and Nuclear chemistry.chemical_element Spodoptera Calcium Biochemistry Cell Line chemistry.chemical_compound Animals Inositol 1 4 5-Trisphosphate Receptors Inositol Calcium Signaling Enzyme Inhibitors Binding site Receptor Molecular Biology Calcium signaling Sulfonamides Binding Sites biology Cell Biology Transfection Recombinant Proteins Rats Cell biology Electrophysiology carbohydrates (lipids) chemistry Calcium-Calmodulin-Dependent Protein Kinases Mutagenesis Site-Directed biology.protein Calcium Channels Chickens Research Article |
| Zdroj: | Biochemical Journal. 365:659-667 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20011789 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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