CHARACTERIZATION OF [H-3] INOSITOL 1,4,5-TRISPHOSPHATE BINDING-SITES IN HUMAN TEMPORAL CORTICAL AND CEREBELLAR MEMBRANES

The characteristics of [H-3]Ins(1,4,5)P3 binding to human temporal cor tical and cerebellar membranes have been determined and compared with the binding to calf cerebellar membranes. Association and dissociation of ligand was very rapid, k, and k-1 values of the order of 7 x 10(7) M-1 min-1 and 0.2 min-1, respectively. K(D) values were 2.7 and 3.5 n M for temporal cortex and cerebellum, respectively. The corresponding B(max) values were 165 and 482 fmol/mg protein. Binding was influenced in a biphasic manner by calcium. The temporal cortical binding was in hibited by Ins(1,4,5)P3 and analogues with the following IC50 values ( nM): Ins(1,4,5)P3 9.5 and 6.2 (two different salts from different sour ces). Ins(2.4.5)P3 42. Ins(1,3,4,5)P4 670, Ins(1,2,5,6)P4 2620, Ins(3, 4,5,6)P4 4300, Ins(1,3,4.5,6)P5 5490, InsP(6)5280. Ins(4.5)P2 2660, In s(I)P 3300, with the IC50 values for Ins(1,5,6)P3, Ins(1,4)P2 and Ins( 4)P being > 25 muM. The IC50 value for heparin was 2.1 mug/ml. A simil ar pattern was seen in the cerebellum. In both tissues, the Hill slope s were near unity for all compounds except Ins(3,4,5,6)P4, where the s lope was 0.4. The calf cerebellum had a similar ligand specificity (al though the potency was generally lower) when values were expressed rel ative to that of Ins(1,4,5)P3, with the possible exception of Ins(1,3, 4,5)P4, which had a greater relative potency. These data would suggest that in the human temporal cortex and cerebellum, [H-3]Ins(1,4,5)P3 b inding sites are expressed in different densities. but have similar pr operties. There may, however, be species differences in the [H-3]Ins(1 ,4.5)P3 recognition site.