Direct photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-[gamma]4-azidoanilide.
Direct photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-[gamma]4-azidoanilide.
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[γ-(32)P]ATP-γ4-叠氮基苯胺直接光亲和标记 Kir6.2。
DOI：
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
K. Ueda
中科院分区：
文献类型：
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作者：
K. Tanabe;S. Tucker;F. Ashcroft;P. Proks;N. Kioka;T. Amachi;K. Ueda
ATP敏感性钾(K(ATP))通道受细胞内ATP和ADP的复合调节。镁ADP的增强作用是通过该通道的磺脲受体亚基SUR来实现的，而ATP的抑制作用似乎是通过成孔亚基Kir6.2来实现的。我们以前报道过Kir6.2可以被8-叠氮基-[伽马-(32)P]ATP直接标记。然而，8-叠氮基-ATP与Kir6.2的结合亲和力较低，可能是由于腺嘌呤的8‘位修饰所致。在这里，我们证明了Kir6.2可以被[Gamma-(32)P]ATP-[Gamma]4-叠氮苯胺([Gamma-(32)P]ATP-AA)直接标记为具有较高亲和力的光亲和物，其中含有一个未修饰的腺嘌呤环。[Gamma-(32)P]ATP-AA对Kir6.2的光亲和标记不受镁离子存在的影响，这与镁离子非依赖的ATP对K(ATP)通道的抑制作用一致。有趣的是，8-叠氮基-三磷酸腺苷(8-叠氮基-三磷酸腺苷)标记的SUR1不是三磷酸腺苷-氨基酸标记的光亲和性体。这些结果确定了SUR1和Kir6.2上核苷酸结合位点结构的关键差异。
ATP-sensitive potassium (K(ATP)) channels are under complex regulation by intracellular ATP and ADP. The potentiatory effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We have previously reported that Kir6.2 can be directly labeled by 8-azido-[gamma-(32)P]ATP. However, the binding affinity of 8-azido-ATP to Kir6.2 was low probably due to modification at 8' position of adenine. Here we demonstrate that Kir6.2 can be directly photoaffinity labeled with higher affinity by [gamma-(32)P]ATP-[gamma]4-azidoanilide ([gamma-(32)P]ATP-AA), containing an unmodified adenine ring. Photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-AA is not affected by the presence of Mg(2+), consistent with Mg(2+)-independent ATP inhibition of K(ATP) channels. Interestingly, SUR1, which can be strongly and specifically photoaffinity labeled by 8-azido-ATP, was not photoaffinity labeled by ATP-AA. These results identify key differences in the structure of the nucleotide binding sites on SUR1 and Kir6.2.