Development of a functional assay for Ca 2+ release activity of IP 3R and expression of an IP 3R gene fragment in the baculovirus-insect cell system

Abstract

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 Ca 2+ channel, the IP 3 receptor (IP 3R), 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 IP 3R function. Agonist-evoked changes in intracellular Ca 2+ levels [Ca 2+] i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP 3R) , with the core of the IP 3R ligand-binding domain from the Drosophila IP 3R, 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 vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP 3R construct and vmlAchR have been used to assay the modulation of IP 3R-mediated Ca 2+ release, by the ligand sink.