Functional properties of recombinant type I and type III inositol 1,4,5- trisphosphate receptor isoforms expressed in COS-7 cells

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.