INOSITOL 1,4,5-TRISPHOSPHATE SLOWLY CONVERTS ITS RECEPTOR TO A STATE OF HIGHER AFFINITY IN SHEEP CEREBELLUM MEMBRANES

Incubation of cerebellar microsomes with D-myo-inositol 1,4,5-trisphos phate (InsP(3)) (0.01-1 mu M), at 4 or 20 degrees C in a cytosolic-lik e medium devoid of Ca2+ and Mg2+, followed by InsP(3) removal, induced an increase in InsP(3) binding determined with 1 nM [H-3]InsP(3). At 20 degrees C, and pH 7.1, maximal stimulation (1.5-2.5-fold) was obtai ned with 1 mu M InsP(3), and the EC(50) was 60 +/- 5 mu M. Several lin es of evidence suggested that the activating site is identical with th e InsP(3) binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a compe titive inhibitor of [H-3]InsP(3) binding, to the preincubation mixture , prevented the activating effect of InsP(3); (iii) the concentration of InsP(3) giving half-maximal activation was close to that giving hal f-maximal InsP(3) binding. The time course of activation was found to be much slower than that of binding. While a t(1/2) less than 0.4 s ha s been measured recently at neutral pH and 20 degrees C for binding of 0.5 nM [H-3]InsP(3) (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L. , Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 26 9, 29642-29649), a 20-s preincubation with 1 mu M InsP(3) was required to half-maximally stimulate binding. Under the present conditions, th e InsP(3)-induced binding increase was only partially reversible. Howe ver, this effect was not blocked by antiproteases suggesting that it d id not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP(3) binding and InsP(3)-dependent activation, w e performed binding experiments on a short period (3 s) to determine t he effect of InsP(3) pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor with out changing the number of binding sites (control: K-D = 107 nM, B-max = 28 pmol/mg of protein; after preincubation with 1 mu M InsP(3): K-D = 53 nM, B-max = 32 pmol/mg of protein). The two states of the recept or bound InsP(3) with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP(3) pretreatment, equilibrium bindin g experiments performed on 10-min incubations revealed an apparent pos itive cooperative behavior (apparent Hill coefficient = 1.6; apparent K-D = 66 nM). These results report a new regulatory process of the Ins P(3) receptor in cerebellum occurring independently of Ca2+ and on a r elatively long time scale.