Wei Y, Bloom P, Gu R, Wang W
Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA.
J Biol Chem. 2000 Jul 7;275(27):20502-7. doi: 10.1074/jbc.M000783200.
Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.
先前的研究表明，蛋白酪氨酸激酶（PTK）活性增加与低钾饮食大鼠皮质集合管（CCD）顶端小电导钾（SK）通道活性下调有关。我们运用膜片钳技术研究蛋白酪氨酸磷酸酶（PTP）在高钾饮食大鼠CCD中SK通道活性调节中的作用。蛋白质印迹分析表明PTP-1D在肾皮质中表达。应用1 μmol苯胂酸（PAO）或1 mmol苄基膦酸（可抑制PTP的试剂）可使通道活性可逆性降低95%。用PAO预处理CCD 30分钟可使平均开放概率（NP(o)）从对照值3.20可逆性降低至0.40。添加1 μmol酪氨酸激酶抑制剂赫曲霉素A对高钾饮食大鼠的CCD通道活性无显著影响。然而，赫曲霉素A消除了PAO的抑制作用，表明PAO的作用是PTK与PTP相互作用的结果。添加布雷菲德菌素A（一种阻断蛋白质从高尔基体复合体向细胞膜转运的试剂）对通道活性无影响。此外，应用微管抑制剂秋水仙碱或微管稳定剂紫杉醇对通道活性也无影响。相比之下，在存在布雷菲德菌素A、秋水仙碱或紫杉醇的情况下，PAO仍可降低通道活性。此外，当肾小管浸泡在含16%蔗糖的浴液中或用伴刀豆球蛋白A处理时，PAO对通道活性的作用消失。我们得出结论，PTP参与SK通道活性的调节，抑制PTP可能促进SK通道的内化。
