INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS IN XENOPUS-LAEVIS OOCYTES - LOCALIZATION AND MODULATION BY CA2+

Inositol 1,4,5-trisphosphate receptors (InsP(3)R) in Xenopus laevis oo cytes were localized and their regulation by Ca2+ was investigated. An tibodies raised against the C-terminal region of the mouse cerebellar InsP(3)R (cAb) cross-reacted with a 255 kD protein in Western blots of Xenopus microsomal membranes. Immunolocalization of this protein in c ryosections of oocytes revealed diffuse staining of the cytoplasm, int ense staining of the sub-plasma membrane region of the animal hemisphe re, and punctate staining in association with the germinal vesicle. In the presence of 40 mu M free Ca2+, isolated oocyte membranes exhibite d a high affinity binding site for Ins1,4,5-P-3 (K-D = 5 nM) and a bin ding capacity of 450 fmol/mg protein. The specific binding capacity of oocyte membranes for [H-3]-lns 1,4,5-P-3 increased as the level of fr ee Ca2+ present in binding assays was raised from < 0.1 nM to 4.0 mu M , with an apparent EC(50) Of 60 nM. Increasing the concentration of fr ee Ba2+ failed to facilitate [H-3]-Ins1,4,5-P-3 binding. Other inosito l phosphates competed for Ins1,4,5-P-3 binding sites with approximate IC50 values of: Ins1,3,4,5-P-4 = 79 nM, Ins2,4,5-P-3 = 455 nM and L-In s1,4,5-P-3 = 20 mu M. In addition, 150 mu g/ml (approximately 12 mu M) heparin displaced 50% of bound [H-3]-Ins1,4,5-P-3, whereas caffeine ( 10 mM) had little effect Functional reconstitution of solubilized InsP (3)Rs into lipid bilayers revealed that Ca2+ was a necessary co-agonis t for activation of the InsP(3)R. When InsP(3) (5 mu M, and Ca2+ (5 mu M) were applied together, conductance steps were observed. InsP(3) or Ca2+ alone had little effect. These results suggest that the subcellu lar organization of InsP(3)Rs and the facilitation of InsP(3) binding and channel opening by Ca2+ contribute to the Ins1,4,5-P-3-mediated Ca 2+ spikes, waves, and oscillations observed in Xenopus oocytes.