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 shor
t duration (3 s), with the use of a perfusion protocol. This procedure prev
ented 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 app
rox. 50 % was measured. Both inhibition and its reversal were complete with
in 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 cha
nging the total number of binding sites. Conversely, increasing the [H-3]In
sP(3) concentration from 30 to 400 nM tripled the IC50 for Ca2+ and decreas
ed the maximal inhibition by 63 %. This is similar to a partial competitive
inhibition between InsP(3) binding and inhibitory Ca2+ binding and is cons
istent with InsP(3) and Ca2+ converting InsP(3) receptor into two different
states with different affinities for these ligands. Mn2+ and Sr2+ also inh
ibited [H-3]InsP(3) binding but were respectively only 1/10 and 1/200 as ef
fective 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 conve
rt cerebellar InsP(3) receptor to a low-affinity state and to inhibit its c
hannel activity. Our results also suggest a mechanism by which InsP(3) coun
teracts this Ca2+-dependent inhibition.