Coquil J F, Picard L, Mauger J P
INSERM U442, Signalisation Cellulaire et Calcium, Université Paris Sud, Bâtiment 443, F-91405 Orsay cedex, France.
Biochem J. 1999 Aug 1;341 ( Pt 3)(Pt 3):697-704. doi: 10.1042/0264-6021:3410697.
We have characterized in detail the Ca(2+)-dependent inhibition of [(3)H]Ins(1,4,5)P(3) ([(3)H]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 Ca(2+) 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. Ca(2+) 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 [(3)H]InsP(3) concentration from 30 to 400 nM tripled the IC(50) for Ca(2+) and decreased the maximal inhibition by 63%. This is similar to a partial competitive inhibition between InsP(3) binding and inhibitory Ca(2+) binding and is consistent with InsP(3) and Ca(2+) converting InsP(3) receptor into two different states with different affinities for these ligands. Mn(2+) and Sr(2+) also inhibited [(3)H]InsP(3) binding but were respectively only 1/10 and 1/200 as effective as Ca(2+). No inhibition was observed with Ba(2+). This selectivity is the same as that previously reported for the inhibitory Ca(2+) site of InsP(3)-induced Ca(2+) flux, suggesting that the same site is used by Ca(2+) 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 Ca(2+)-dependent inhibition.
我们使用灌注方案，在短时间内（3秒）详细表征了Ca(2+)对[(3)H]肌醇-1,4,5-三磷酸（[(3)H]InsP(3)）与绵羊小脑微粒体结合的依赖性抑制作用。该方法避免了此前在该Ca(2+)效应研究中发现的假象。在pH 7.1和20摄氏度的类似胞质溶胶的介质中，测得最大抑制率约为50%。抑制及其逆转在3秒内完成。Ca(2+)使受体对InsP(3)的亲和力降低约50%（在pCa 9时K(d)为146±24 nM，在pCa 5.3时为321±56 nM），而不改变结合位点的总数。相反，将[(3)H]InsP(3)浓度从30 nM增加到400 nM，使Ca(2+)的IC(50)增加两倍，并使最大抑制率降低63%。这类似于InsP(3)结合与抑制性Ca(2+)结合之间的部分竞争性抑制，并且与InsP(3)和Ca(2+)将InsP(3)受体转化为对这些配体具有不同亲和力的两种不同状态一致。Mn(2+)和Sr(2+)也抑制[(3)H]InsP(3)结合，但效力分别仅为Ca(2+)的1/10和1/200。未观察到Ba(2+)有抑制作用。这种选择性与先前报道的InsP(3)诱导的Ca(2+)通量的抑制性Ca(2+)位点相同，表明Ca(2+)利用相同的位点将小脑InsP(3)受体转化为低亲和力状态并抑制其通道活性。我们的结果还提示了一种InsP(3)抵消这种Ca(2+)依赖性抑制的机制。
