Effect of inositol tetrakisphosphates on inositol 1,4,5-trisphosphate binding to rat liver microsomes

lnositol 1,4,5-trisphosphate [Ins( 1,4,5)P3] is produced by the phosphodiesteratic cleavage of the membrane lipid phosphatidylinositol4,S-bisphosphate in many cell types upon stimulation. It is thought to elevate intracellular free calcium ion concentration by stimulating release from a non-mitochondrial storage site [ 11. The mechanism by which Ins( 1,4,5)P3 exerts its action is largely unknown, but is presumed to involve interaction with a receptor site on an intracellular membrane (21. We have investigated the characteristics of binding of "P-labelled Ins( 1,4,5)P3 to fresh rat liver microsomes. The ['2P]Ins( 1,4,5)P3 binding was found to be rapid and saturable. The half-time for the association was approx. 2 min at 4"C, but 30 min incubation was required for binding to achieve equilibrium. The half-time for dissociation was approximately 5 min. Binding was saturable, and greater than 90% of total binding could be displaced by excess added non-radioactive Ins( 1 ,4,5)P3 (8.57 p ~ ) . Scatchard analysis revealed two binding sites. The high affinity site had a K , of 3.9 n M and B,, , of 32 fmol/mg protein. The low affinity site had a K , of 50 nM and B,,,, of 0.224 pmol/mg protein. We went on to investigate the ability of various inositol derivatives to displace Ins( 1 ,4,5)P3 from the high affinity site by incubating microsomal membranes with 0.185 nM["P]Ins( l,4,5)P3 and various concentrations of the added compounds under equilibrium binding conditions. These included two isomers of inositol tetrakisphosphate (InsP,): Ins( 1,3,4,5)P4 which is produced in mammalian cells by the action of a 3' kinase on Ins( 1,4,5)P3 [3] and Ins( 1,4,5,6)P4 which is present in avian erythrocytes [4]. As can be seen in Fig. 1, several substances were able to compete with high affinity Ins( 1,4,S)P3 binding. In order of potency with the concentration giving 50% displacement of specific binding in parentheses these were: inositol 2,4,5-trisphosphate [lns(2,4,5)P3] (56 n M ) , Ins( 1,3,4,5)P4 (1 10 nM) , inositol 4,sbisphosphate [lns(4,5)P2] (1.5 p ~ ) , Ins( 1,4,5,6)P4 (10.5 ,UM by extrapolation). For the sake of clarity, the following were not included in the Figure: inositol4-phosphate [Ins(4)P] (23 ,UM by extrapolation) and inositol 1,4-bisphosphate [Ins( 1,4)P2] (estimated at > 50 ,uM). Inositol 1-phosphate, inositol 2-phosphate and inositol hexasulphate were unable to compete for Ins( 1,4,5)P3 binding even up to 50 PM. On the basis of these results it is possible to draw some tentative conclusions on the structure-activity relationships for this high affinity binding site on rat liver microsomes. By removing one phosphate from Ins( 1,4,5)P3 it is possible to generate three different inositol bisphosphate (InsP,) isomers. We have only tested two of these as we could not obtain Ins( 1,5)P2. The relative potency of lns(4,5)P2 versus