Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels

The mechanism for coupling between Ca2+ stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxy-diphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 8-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr2+ entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trp1 mutant flies were all inhibited by 2-APB, This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. Ca-45(2+) flux analysis revealed that although DT40 wildtype cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca2+ release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca2+ store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC50) were identical. The results indicate that (a) the action of 2-APB on Ca2+ entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.