Liou H H, Zhou S S, Huang C L
Department of Medicine, Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, TX 75235-8856, USA.
Proc Natl Acad Sci U S A. 1999 May 11;96(10):5820-5. doi: 10.1073/pnas.96.10.5820.
ROMK inward-rectifier K+ channels control renal K+ secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Having found that direct interaction with membrane phosphatidylinositol 4, 5-bisphosphate (PIP2) is essential for channel activation, we investigate here the role of PIP2 in regulation of ROMK1 by PKA. By using adenosine-5'-[gamma-thio]triphosphate) (ATP[gammaS]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP2. Rather, phosphorylation by PKA + ATP[gammaS] lowers the concentration of PIP2 necessary for activation of the channels. In solution-binding assays, anti-PIP2 antibodies bind PIP2 and prevent PIP2-channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 for inhibition by the antibodies, indicating an enhanced interaction between PIP2 and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP2 interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP2-channel interaction.
ROMK内向整流钾通道控制肾脏钾分泌。ROMK的活性受蛋白激酶A（PKA）调节，但其调节的分子机制尚不清楚。由于发现与膜磷脂酰肌醇4,5-二磷酸（PIP2）的直接相互作用对通道激活至关重要，我们在此研究PIP2在PKA对ROMK1调节中的作用。通过使用腺苷-5'-[γ-硫代]三磷酸（ATP[γS]）作为底物，我们发现PKA不会直接激活不含PIP2的膜中的ROMK1通道。相反，PKA + ATP[γS]的磷酸化降低了激活通道所需的PIP2浓度。在溶液结合试验中，抗PIP2抗体结合PIP2并阻止PIP2与通道的相互作用。在内外翻膜片中，抗体抑制通道的活性。然后PKA处理降低了ROMK1对抗体抑制的敏感性，表明PIP2与磷酸化通道之间的相互作用增强。相反，ROMK1中PKA磷酸化位点的突变降低了PIP2与通道的相互作用。因此，PKA通过增强PIP2与通道的相互作用来激活ROMK1通道。
