Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+

We have characterized in detail the Ca2+-dependent inhibition of [H-3]Ins(1,4,5)P-3 ([H-3]InsP(3)) binding to sheep cerebellar microsomes, over a short duration (3 s), with the use of a perfusion protocol. This procedure prevented artifacts previously identified in studies of this Ca2+ effect. In a cytosol-like medium at pH 7.1 and 20 degrees C, a maximal inhibition of approx. 50 % was measured. Both inhibition and its reversal were complete within 3 s. Ca2+ decreased the affinity of the receptor for InsP(3) by approx. 50 % (K-d 146 +/- 24 nM at pCa 9 and 321 +/- 56 nM at pCa 5.3), without changing the total number of binding sites. Conversely, increasing the [H-3]InsP(3) concentration from 30 to 400 nM tripled the IC50 for Ca2+ and decreased the maximal inhibition by 63 %. This is similar to a partial competitive inhibition between InsP(3) binding and inhibitory Ca2+ binding and is consistent with InsP(3) and Ca2+ converting InsP(3) receptor into two different states with different affinities for these ligands. Mn2+ and Sr2+ also inhibited [H-3]InsP(3) binding but were respectively only 1/10 and 1/200 as effective as Ca2+. No inhibition was observed with Ba2+. This selectivity is the same as that previously reported for the inhibitory Ca2+ site of InsP(3)-induced Ca2+ flux, suggesting that the same site is used by Ca2+ to convert cerebellar InsP(3) receptor to a low-affinity state and to inhibit its channel activity. Our results also suggest a mechanism by which InsP(3) counteracts this Ca2+-dependent inhibition.