Moral Z, Dong K, Wei Y, Sterling H, Deng H, Ali S, Gu R, Huang X Y, Hebert S C, Giebisch G, Wang W H
Department of Pharmacology, New York Medical College, Valhalla, New York 10595, USA.
J Biol Chem. 2001 Mar 9;276(10):7156-63. doi: 10.1074/jbc.M008671200. Epub 2000 Dec 12.
We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.
我们运用双电极电压钳技术和膜片钳技术，研究了在共表达ROMK1和cSrc的卵母细胞中，蛋白酪氨酸磷酸酶（PTP）和蛋白酪氨酸激酶（PTK）对ROMK1通道的调节作用。蛋白质印迹分析检测到卵母细胞中存在内源性PTP-1D同工型。添加苯砷氧化物（PAO），一种PTP抑制剂，可使共注射ROMK1和cSrc的卵母细胞中的钾离子电流可逆性降低55%。相比之下，PAO对单独注射ROMK1的卵母细胞中的钾离子电流无显著影响。此外，应用酪氨酸激酶抑制剂赫曲霉素A可使共表达ROMK1和cSrc的卵母细胞中的钾离子电流增加120%，并完全消除PAO的作用。在表达ROMK1突变体R1Y337A（其中第337位酪氨酸残基突变为丙氨酸）的卵母细胞中，赫曲霉素A和PAO没有作用。然而，添加外源性cSrc对外翻膜片中ROMK1通道的活性无显著影响。此外，用20%蔗糖和250μg/ml伴刀豆球蛋白A处理卵母细胞，这两种试剂可抑制ROMK1通道的内吞作用，完全消除了PAO的作用。此外，用微管抑制剂秋水仙碱或使微管固定的试剂紫杉醇预处理卵母细胞后，赫曲霉素A的作用消失。我们得出结论，PTP和PTK在调节ROMK1通道中起重要作用。抑制PTP会增加ROMK1通道的内化，而阻断PTK则会刺激ROMK1通道的插入。
