D. Boehning et Sk. Joseph, Functional properties of recombinant type I and type III inositol 1,4,5-trisphosphate receptor isoforms expressed in COS-7 cells, J BIOL CHEM, 275(28), 2000, pp. 21492-21499
Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are ubiquitous intracellul
ar Ca2+ release channels whose functional characterization by transfection
has proved difficult due to the background contribution of endogenous chann
els. In order to develop a functional assay to measure recombinant channels
, we transiently transfected the rat type I IP3R into COS-7 cells. Saponin-
permeabilized 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 mu M) but no enhancement at subsaturating [IP3] (300 nM). However,
cotransfection of the IP3R and human sarco/endoplasmic reticulum ATPase (S
ERCA)-2b ATPase cDNA resulted in 60 and 110% increases in Ca2+ release at s
ubsaturating and saturating doses of IP3, respectively. IP3 or adenophostin
A failed to release Ca-45(2+) from microsomal vesicles prepared from cells
expressing either type I IP3R or SERCA cDNAs alone. However, microsomal ve
sicles prepared from cells doubly transfected with IP3R and SERCA cDNAs rel
eased 33.0 +/- 0.04% of the A23187-sensitive pool within 30 a of 1 mu M ade
nophostin A addition. Similarly, the initial rate of Ca-45(2+) influx into
oxalate-loaded microsomal vesicles was inhibited by IP3 only when the micro
somes were prepared from COS cells doubly transfected with SERCA-2b and IP3
R DNA. The absence of a functional contribution from endogenous IP(3)Rs has
enabled the use of this assay to measure the Ca2+ sensitivities of IP3-med
iated Ca-45(2+) fluxes through recombinant neuronal type I (SII(+)), periph
eral type I (SII(-)), and type III IP(3)Rs. 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 Ca-45(2+) fluxes, whereas the conservative substitution D2550E wa
s without effect. This assay therefore provides a useful tool for studying
the regulatory properties of individual IP3R isoforms as well as for screen
ing pore mutations prior to more detailed electrophysiological analyses.