Two inositol 1,4,5-trisphosphate binding sites in rat basophilic leukemia cells: relationship between receptor occupancy and calcium release.
Quantal calcium release is a novel paradigm for second messenger signal transduction which provides spatial and temporal control of calcium release from intracellular stores by inositol 1,4,5-trisphosphate (InsP3). We have proposed a mechanism to account for this phenomenon [Kindman, L. A., & Meyer, T. (1993) Biochemistry 32, 1270-1277], which hypothesized the existence of five channels, each with a different affinity for InsP3. As a direct test of this hypothesis, InsP3 binding to microsomes from RBL cells was examined under conditions similar to those used for calcium release. Scatchard analyses performed under a variety of conditions indicates the presence of high affinity (KD = 0.9 +/- 0.3 nM) and low affinity (KD = 47 +/- 5 nM) InsP3 binding sites. The low affinity sites are more prevalent, constituting 82 +/- 5% of the total. Both sites are identified in the presence and absence of MgATP. Moreover, both sites are selective for InsP3 over InsP4, through high concentrations of InsP4 displace InsP3 from each site (with inhibition constants of 16 and 267 nM InsP4, respectively). The relative abundance of the two InsP3 binding sites is Ca2+ dependent. An increase in Ca2+ from 0.1 to 0.5 microM results in the apparent conversion of a portion of the low affinity sites into high affinity sites into high affinity sites. Ca2+ (0.5 microM) also increased the KD of the low affinity InsP3 binding site. Given the presence of both high and low affinity InsP3 binding sites, two simple mathematical models describing both the kinetics of calcium release and quantal calcium release from RBL cells were developed.(ABSTRACT TRUNCATED AT 250 WORDS)
Watras
J
J
University of Connecticut Health Center, Farmington 06032.
Moraru
I
I
Costa
D J
DJ
Kindman
L A
LA
D019794:2,3-Diphosphoglycerate; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism*; D008433:Mathematics; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D004163:Diphosphoglyceric Acids; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; C081655:inositol-1,3,4,5-tetrakisphosphate; D019794:2,3-Diphosphoglycerate; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium