Regulation of Ca2+-dependent Cl- conductance in a human colonic epithelial cell line (T84):: Cross-talk between Ins(3,4,5,6)P4 and protein phosphatases
Regulation of Ca2+-dependent Cl- conductance in a human colonic epithelial cell line (T84):: Cross-talk between Ins(3,4,5,6)P4 and protein phosphatases
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DOI：
10.1111/j.1469-7793.1998.661bj.x
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发表时间：
1998-08-01
影响因子：
5.5
通讯作者：
Shears, SB
中科院分区：
文献类型：
--
作者：
Xie, WW;Solomons, KRH;Shears, SB
1.我们研究了3，4，5，6-四磷酸肌醇（Ins（3，4，5，6）P-4）对T-84结肠上皮细胞全细胞氯电流的调节。利用（a）微囊藻毒素和冈田酸抑制丝氨酸/苏氨酸蛋白磷酸酶和（B）新的功能性四磷酸类似物1，2-双脱氧-1，2-双氟-Ins（3，4，5，6）P-4（即F-2-Ins（3，4，5，6）P-4）获得了新的信息。钙调蛋白依赖性蛋白激酶II（CaMKII）增加氯电流20倍。该电流（I-Cl，I-CaMK）持续7 +/- 1.2 min，然后失活或下降约60%。冈田酸防止了这种下降，因此I-Cl，I-CaMK保持在其最大值附近大于或等于14.3 +/- 0.6分钟。F-2-Ins（3，4，5，6）P-4立体特异性地抑制I-Cl，I-CaMK（IC 50 = 100 μ M），因为其对映体F-2-Ins（1，4，5，6）P-4在小于或等于500 μ M时没有作用。剂量-反应数据（Hill系数= 1.3）显示F-2-Ins（3，4，5，6）P-4仅模拟Ins（3，4，5，6）P-4抑制的非合作相，而非合作相.通过冈田酸（IC 50 = 1.5 nM）和微囊藻毒素（IC 50 = 0.15 nM）阻止Ins（3，4，5，6）P阻断I-Cl，I-CaMK;这些数据导致新的结论，即原位蛋白磷酸酶活性对于Ins（3，4，5，6）P发挥功能是必需的。IC值表明需要一种以上的磷酸酶。其中之一可能是PP 1，因为冈田酸和微囊藻毒素抑制F-2- Ins（3，4，5，6）P-4依赖性电流阻断，IC 50值分别为70 nM和0.15 nM。
1. We have studied the regulation of whole-cell chloride current in T-84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P-4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1,2-bisdeoxy-1,2-bisfluoro-Ins(3,4,5,6)P-4 (i.e. F-2-Ins(3,4,5,6)P-4).2. Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (I-Cl,I-CaMK) continued for 7 +/- 1.2 min before its deactivation, or running down, by approximately 60%. This run-down was prevented by okadaic acid, whereupon I-Cl,I-CaMK remained near its maximum value for greater than or equal to 14.3 +/- 0.6 min.3. F-2-Ins(3,4,5,6)P-4 inhibited I-Cl,I-CaMK (IC50 = 100 mu M) stereo-specifically since its enantiomer, F-2-Ins(1,4,5,6)P-4 had no effect at less than or equal to 500 mu M. Dose-response data (Hill coefficient = 1.3) showed that F-2-Ins(3,4,5,6)P-4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P-4, and not the co-operative phase.4. Ins(3,4,5,6)P, was prevented from blocking I-Cl,I-CaMK by okadaic acid (IC50 = 1.5 nM) and microcystin (IC50 = 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P, to function. The IC,, values indicate that more than one species of phosphatase was required. One of these may be PP1, since F-2- Ins(3,4,5,6)P-4-dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.