Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system.
Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP3), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca2+ channel, the IP3 receptor (IP3R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf (S. frugiperda) cell system that can be used to look at IP3R function. Agonist-evoked changes in intracellular Ca2+ levels [Ca2+]i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vm1AchR). Furthermore, we have constructed a recombinant BV (vIP3R), with the core of the IP3R ligand-binding domain from the Drosophila IP3R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vm1AchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP3R construct and vm1AchR have been used to assay the modulation of IP3R-mediated Ca2+ release, by the ligand sink.
Raghu
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P
National Center for Biological Sciences, TIFR Center, IISc Campus, Bangalore, India.
Habib
S
S
Hasnain
S E
SE
Hasan
G
G
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D004330:Drosophila / Q000235:genetics; D005799:Genes, Dominant; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010641:Phenotype; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D004247:DNA; D002118:Calcium