Finn A L, Gaido M L, Dillard M
Department of Physiology, School of Medicine, University of North Carolina, Chapel Hill 27599.
Mol Cell Biochem. 1992 Sep 8;114(1-2):21-6. doi: 10.1007/BF00240293.
We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M(r) 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (PCl/PNa = 20.2; PNa/PK = 1) whose unit conductance is 62.4 +/- 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01-1.0 microM). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is 'intracellular', (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.
我们使用了一种单克隆抗体（MAb E12），它是针对经茶碱处理的美西螈胆囊上皮细胞产生的几种此类抗体之一，通过免疫亲和柱和快速蛋白质液相色谱法来分离一种氯离子通道蛋白。这种蛋白质（相对分子质量219,000）已被重组到平面脂质双分子层中，在其中它表现为一种氯离子选择性通道（氯离子通透率/钠离子通透率 = 20.2；钠离子通透率/钾离子通透率 = 1），其单位电导为62.4±4.6皮西门子。添加到反侧的抗体（顺侧无影响）会使通道开放概率降至几乎为零，但对单通道的电导或选择性没有影响。为了测试磷酸化在天然通道活性中的作用，我们研究了蛋白磷酸酶抑制剂冈田酸（OA）对完整胆囊的影响，结果表明，经茶碱处理而开放并被抗体关闭的通道可被OA（0.01 - 1.0微摩尔）可逆地重新开放。将蛋白磷酸酶2A（PP - 2A）的催化亚基添加到含有重组氯离子通道的双分子层的顺侧，会导致通道在延迟后关闭，随后添加ATP和环磷酸腺苷依赖性蛋白激酶（PKA）的催化亚基会使通道立即重新开放。这些数据表明：（a）这种氯离子通道蛋白以定向方式插入双分子层，使得顺侧为“细胞内”侧；（b）纯化的通道蛋白被磷酸化；（c）来自细胞侧的门控由通道蛋白的直接磷酸化和去磷酸化控制。
