Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitous intracellular Ca2+ release channels whose functional characterization by transfection has proved difficult due to the background contribution of endogenous channels. In order to develop a functional assay to measure recombinant channels, we transiently transfected the rat type I IP3R into COS-7 cells. Saponinpermeabilized COS cells transfected with type I IP3R showed a 50% increase in inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release at saturating [IP3] (10 μM) but no enhancement at subsaturating [IP3] (300 nM). However, cotransfection of the IP3R and human sarco/endoplasmic reticulum ATPase (SERCA)-2b ATPase cDNA resulted in 60 and 110% increases in Ca2+ release at subsaturating and saturating doses of IP3, respectively. IP3 or adenophostin A failed to release 45Ca2+ from microsomal vesicles prepared from cells expressing either type I IP3R or SERCA cDNAs alone. However, microsomal vesicles prepared from cells doubly transfected with IP3R and SERCA cDNAs released 33.0 ± 0.04% of the A23187- sensitive pool within 30 s of 1 μM adenophostin A addition. Similarly, the initial rate of 45Ca2+ influx into oxalate-loaded microsomal vesicles was inhibited by IP3 only when the microsomes were prepared from COS cells doubly transfected with SERCA-2b and IP3R DNA. The absence of a functional contribution from endogenous IP3Rs has enabled the use of this assay to measure the Ca2+ sensitivities of IP3-mediated 45Ca2+ fluxes through recombinant neuronal type I (SII(+)), peripheral type I (SII(-)), and type III IP3Rs. All three channels displayed a biphasic dependence upon [Ca2+](cyt). Introduction of mutations D2550A and D2550N in the putative pore-forming region of the type I IP3R inhibited IP3-mediated 45Ca2+ fluxes, whereas the conservative substitution D2550E was without effect. This assay therefore provides a useful tool for studying the regulatory properties of individual IP3R isoforms as well as for screening pore mutations prior to more detailed electrophysiological analyses.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology