Ali S, Chen X, Lu M, Xu J Z, Lerea K M, Hebert S C, Wang W H
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):10274-8. doi: 10.1073/pnas.95.17.10274.
In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.
在本研究中，我们运用双电极电压钳和膜片钳技术，研究了福斯高林和环磷酸腺苷（cAMP）对ROMK1通道的影响，据信该通道是肾脏中的天然钾离子分泌通道。在10分钟内添加1微摩尔福斯高林或100微摩尔8-溴环磷酸腺苷，对非洲爪蟾卵母细胞中表达的ROMK1通道电流没有显著影响。相比之下，当ROMK1通道与从神经组织克隆的A激酶锚定蛋白AKAP79共表达时，在3分钟内施加1微摩尔福斯高林可使全细胞钾离子电流显著增加35%。有两条证据表明福斯高林的作用是由cAMP依赖性途径介导的：（i）添加100微摩尔8-溴环磷酸腺苷可模拟福斯高林的作用；（ii）福斯高林和cAMP的作用并非相加的。cAMP发挥作用需要AKAP这一点，在共表达ROMK1和AKAP79的卵母细胞中，添加三磷酸腺苷（100微摩尔）和cAMP（100微摩尔）可恢复内面向外膜片中逐渐衰减的ROMK1通道活性，但在仅表达ROMK1的卵母细胞中则不能恢复，这一实验进一步证明了这一点。此外，当我们在覆盖分析中使用II型蛋白激酶A的调节亚基RII时，我们在从肾皮质获得的膜中鉴定出一种RII结合蛋白，但在卵母细胞膜中未鉴定出。这表明ROMK1通道对福斯高林和cAMP不敏感是由于缺乏AKAP。我们得出结论，AKAP可能是介导蛋白激酶A对肾脏中ROMK通道作用的关键成分。
