Coquil J F, Mauger J P, Claret M
Unité de Recherche U.274, INSERM, Université Paris-Sud, 91405 Orsay Cedex, France.
J Biol Chem. 1996 Feb 16;271(7):3568-74. doi: 10.1074/jbc.271.7.3568.
Incubation of cerebellar microsomes with d-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01 1 microM), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nm [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5 2. 5-fold) was obtained with 1 mum InsP3, and the EC50 was 60 +/- 5 nm. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nm [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 microM InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nm, Bmax = 28 pmol/mg of protein; after preincubation with 1 microM InsP3: KD = 53 nm, Bmax = 32 pmol/mg of protein). The two states of the receptor bound InsP3 with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP3 pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent KD = 66 nm). These results report a new regulatory process of the InsP3 receptor in cerebellum occurring independently of Ca2+ and on a relatively long time scale.
在无Ca2+和Mg2+的胞质样介质中，于4℃或20℃将小脑微粒体与d - 肌醇1,4,5 - 三磷酸（InsP3）（0.01 - 1μM）温育，随后去除InsP3，结果显示用1 nM [3H]InsP3测定时InsP3结合增加。在20℃和pH 7.1条件下，用1μM InsP3可获得最大刺激（1.5 - 2.5倍），半数有效浓度（EC50）为60±5 nM。多条证据表明激活位点与InsP3结合位点相同：（i）激活和结合表现出相同的肌醇磷酸特异性；（ii）向预温育混合物中添加十钒酸盐（[3H]InsP3结合的竞争性抑制剂）可阻止InsP3的激活作用；（iii）产生半数最大激活的InsP3浓度接近产生半数最大InsP3结合的浓度。发现激活的时间进程比结合的时间进程慢得多。虽然最近在中性pH和20℃条件下测得0.5 nM [3H]InsP3结合的半衰期小于0.4秒（Hannaert - Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642 - 29649），但用1μM InsP3预温育20秒才能使结合达到半数最大刺激。在当前条件下，InsP3诱导的结合增加只是部分可逆的。然而，这种效应未被抗蛋白酶阻断，表明它不涉及蛋白水解。利用InsP3结合和InsP3依赖性激活动力学的显著差异，我们在短时间（3秒）内进行结合实验以确定InsP3预处理对结合参数的影响。数据表明这种处理增加了受体的亲和力，而不改变结合位点的数量（对照：KD = 107 nM，Bmax = 28 pmol/mg蛋白质；用1μM InsP3预温育后：KD = 53 nM，Bmax = 32 pmol/mg蛋白质）。在3秒的时间尺度上，受体的两种状态结合InsP3时的希尔系数接近1。与InsP3预处理的效果一致，在10分钟温育后进行的平衡结合实验显示出明显的正协同行为（表观希尔系数 = 1.6；表观KD = 66 nM）。这些结果报道了小脑InsP3受体一种新的调节过程，该过程独立于Ca2+且发生在相对较长的时间尺度上。
