Regulation of WNK1 kinase by extracellular potassium
Regulation of WNK1 kinase by extracellular potassium
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DOI：
10.1007/s10157-010-0378-9
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发表时间：
2011-04-01
影响因子：
2.3
通讯作者：
Uchida, Shinichi
中科院分区：
文献类型：
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作者：
Naito, Shotaro;Ohta, Akihito;Uchida, Shinichi
WNK激酶基因突变被认为是一种遗传性高血压病-假性低醛固酮增多症II型的原因；然而，对WNK激酶的调节知之甚少。在本研究中，我们以非等渗条件为线索，通过底物OSR1的磷酸化来监测COS7细胞内源性WNK激酶的活性，发现WNK1是COS7细胞中主要的WNK激酶。与高渗液引起的WNK1活性一过性升高相反，低渗液使OSR1的磷酸化水平持续增加24 h，提示低渗液中含有持续刺激WNK1活性的信号。为了识别该信号，进行了离子置换实验。令人惊讶的是，即使是低Cl-或低K+的等渗介质也能增加OSR1的磷酸化，与低渗介质一样。此外，低渗液中WNK1的激活可被奎宁(500 MU M)完全阻断，但不能被5-硝基-2-(3-苯丙氨基)苯甲酸(100 MU M)所阻断，这种抑制作用与抑制Rb-86(+)(=K+)外排密切相关，而与抑制I-125(-)(=Cl-)外排无关。这些结果表明，K+而不是低渗或低氯可能是WNK1激活的重要调节因素。最后，我们证实了生理范围内的高K+和低K+介质分别降低和提高了WNK1的活性，胞外K+是WNK1活性的重要调节因子。
Mutations of WNK kinase genes were identified as the cause of a hereditary hypertensive disease, pseudohypoaldosteronism type II; however, little is known about the regulation of WNK kinases. In the present study, we focused on anisosmotic conditions as the initial clues for clarifying a stimulating factor for WNK kinase activity.Endogenous WNK kinase activity in COS7 cells was monitored by the phosphorylation of its substrate, OSR1.Knockdown experiments revealed that WNK1 was a major WNK kinase in COS7 cells. In contrast to the transient increase in WNK1 activity caused by hypertonic medium, hypotonic medium increased the phosphorylation of OSR1 for 24 h, suggesting that the hypotonic medium included a signal for continuously stimulating WNK1 kinase activity. To identify the signal, ion substitution experiments were performed. Surprisingly, even isotonic media with low Cl- or low K+ was found to increase OSR1 phosphorylation as well as the hypotonic medium. Furthermore, WNK1 activation by the hypotonic medium was completely blocked by quinine (500 mu M) but not by 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 mu M), and this inhibition was closely correlated with the inhibition of Rb-86(+) (=K+) efflux but not with the inhibition of I-125(-) (=Cl-) efflux. These results suggest that K+, rather than hypotonicity or low Cl-, may be an important regulator for WNK1 activation. Finally, we confirmed that high K+ and low K+ media under the physiological range decreased and increased WNK1 activity, respectively.Extracellular K+ is an important regulator of WNK1 kinase activity.