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 (Ip(3)), 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 vIP(3)R construct and vm1AchR have been used to assay the modulation of IP3R-mediated Ca2+ release, by the ligand sink.