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 (IP(3)Rs) are ubiquitous intracellular Ca(2+) 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 IP(3)R into COS-7 cells. Saponin-permeabilized COS cells transfected with type I IP(3)R showed a 50% increase in inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release at saturating [IP(3)] (10 micrometer) but no enhancement at subsaturating [IP(3)] (300 nm). However, cotransfection of the IP(3)R and human sarco/endoplasmic reticulum ATPase (SERCA)-2b ATPase cDNA resulted in 60 and 110% increases in Ca(2+) release at subsaturating and saturating doses of IP(3), respectively. IP(3) or adenophostin A failed to release (45)Ca(2+) from microsomal vesicles prepared from cells expressing either type I IP(3)R or SERCA cDNAs alone. However, microsomal vesicles prepared from cells doubly transfected with IP(3)R and SERCA cDNAs released 33.0 +/- 0.04% of the A23187-sensitive pool within 30 s of 1 micrometer adenophostin A addition. Similarly, the initial rate of (45)Ca(2+) influx into oxalate-loaded microsomal vesicles was inhibited by IP(3) only when the microsomes were prepared from COS cells doubly transfected with SERCA-2b and IP(3)R DNA. The absence of a functional contribution from endogenous IP(3)Rs has enabled the use of this assay to measure the Ca(2+) sensitivities of IP(3)-mediated (45)Ca(2+) fluxes through recombinant neuronal type I (SII(+)), peripheral type I (SII(-)), and type III IP(3)Rs. All three channels displayed a biphasic dependence upon [Ca(2+)](cyt). Introduction of mutations D2550A and D2550N in the putative pore-forming region of the type I IP(3)R inhibited IP(3)-mediated (45)Ca(2+) fluxes, whereas the conservative substitution D2550E was without effect. This assay therefore provides a useful tool for studying the regulatory properties of individual IP(3)R isoforms as well as for screening pore mutations prior to more detailed electrophysiological analyses.
Boehning
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Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107, USA.
Joseph
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SK
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000502:physiology; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; C085029:adenophostin A; D000001:Calcimycin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D000241:Adenosine; D002118:Calcium