Inositol 1,4,5-trisphosphate receptors in Xenopus laevis oocytes: localization and modulation by Ca2+.
Inositol 1,4,5-trisphosphate receptors (InsP3R) in Xenopus laevis oocytes were localized and their regulation by Ca2+ was investigated. Antibodies raised against the C-terminal region of the mouse cerebellar InsP3R (cAb) cross-reacted with a 255 kD protein in Western blots of Xenopus microsomal membranes. Immunolocalization of this protein in cryosections of oocytes revealed diffuse staining of the cytoplasm, intense staining of the sub-plasma membrane region of the animal hemisphere, and punctate staining in association with the germinal vesicle. In the presence of 40 microM free Ca2+, isolated oocyte membranes exhibited a high affinity binding site for Ins 1,4,5-P3 (KD = 5nM) and a binding capacity of 450 fmol/mg protein. The specific binding capacity of oocyte membranes for [3H]-Ins 1,4,5-P3 increased as the level of free Ca2+ present in binding assays was raised from < 0.1 nM to 4.0 microM, with an apparent EC50 of 60 nM. Increasing the concentration of free Ba2+ failed to facilitate [3H]-Ins1,4,5-P3 binding. Other inositol phosphates competed for Ins1,4,5-P3 binding sites with approximate IC50 values of: Ins1,3,4,5-P4 = 79 nM, Ins2,4,5-P3 = 455 nM and L-Ins1,4,5-P3 = 20 microM. In addition, 150 micrograms/ml (approximately 12 microM) heparin displaced 50% of bound [3H]-Ins1,4,5-P3, whereas caffeine (10 mM) had little effect. Functional reconstitution of solubilized InsP3Rs into lipid bilayers revealed that Ca2+ was a necessary co-agonist for activation of the InsP3R. When InsP3 (5 microM) and Ca2+ (5 microM) were applied together, conductance steps were observed. InsP3 or Ca2+ alone had little effect. These results suggest that the subcellular organization of InsP3Rs and the facilitation of InsP3 binding and channel opening by Ca2+ contribute to the Ins1,4,5-P3-mediated Ca2+ spikes, waves, and oscillations observed in Xenopus oocytes.
Callamaras
N
N
Department of Psychobiology, University of California, Irvine.
Parker
I
I
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D015220:Calcium Channels / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006493:Heparin / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009865:Oocytes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D014982:Xenopus laevis
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D002110:Caffeine; D006493:Heparin; D002118:Calcium