Direct Photoaffinity Labeling of the Kir6.2 Subunit of the ATP-sensitive K+ Channel by 8-Azido-ATP*
Direct Photoaffinity Labeling of the Kir6.2 Subunit of the ATP-sensitive K+ Channel by 8-Azido-ATP*
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通过 8-叠氮基-ATP* 直接光亲和标记 ATP 敏感 K 通道的 Kir6.2 亚基
DOI：
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发表时间：
1999
影响因子：
4.8
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;M. Matsuo;P. Proks;F. Ashcroft;S. Seino;T. Amachi;K. Ueda
ATP敏感性钾通道受细胞内ATP和ADP的复杂调节。MgADP的增强作用是由通道的磺酰脲受体亚基SUR赋予的，而ATP的抑制作用似乎是通过孔形成亚基Kir6.2介导的。我们通过分析ATP（8-叠氮基-[γ-32 P]ATP）的光亲和类似物与瞬时表达Kir6.2的COS-7细胞膜的结合，确定ATP是否直接与Kir6.2亚基上的结合位点相互作用以介导通道抑制。我们证明Kir6.2可以被8-叠氮基-[γ-32 P]ATP直接标记，但不被ATP抑制的相关亚基Kir4.1不被标记。100 μm ATP使Kir6.2的光亲和标记减少约50%。此外，Kir6.2的NH 2末端（R50 G）和COOH末端（K185 Q）的突变均显示出降低ATP对Kir6.2通道活性的抑制作用，使光亲和标记降低> 50%。这些结果表明，ATP直接结合Kir6.2和NH 2-和COOH-末端的细胞内结构域可能会影响ATP的结合。
ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating 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 determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[γ-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[γ-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 μm ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.