Mapping the Gβγ-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel
Mapping the Gβγ-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel
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DOI：
10.1074/jbc.m304518200
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发表时间：
2003-08-01
影响因子：
4.8
通讯作者：
Dascal, N
中科院分区：
文献类型：
--
作者：
Ivanina, T;Rishal, I;Dascal, N
G 蛋白激活的 K+ 通道（Kir3 或 GIRK）通过直接结合 Gbetagamma 来激活。尚未明确绘制普遍存在的 GIRK1 (Kir3.1) 亚基中 Gbetagamma 的结合位点，并且尚未研究神经元 GIRK2 (Kir3.2) 亚基中 Gbetagamma 的结合。我们使用两种方法验证并扩展了 GIRK1 中 Gbetagamma 结合位点的图谱：Gbetagamma 与 GIRK 亚基片段直接结合（下拉），以及这些片段与 Galpha(i1) 亚基竞争与 Gbetagamma 的结合。我们还绘制了 GIRK2 中的 Gbetagamma 结合位点。在两个亚基中，N 末端均结合 Gbetagamma。在C末端，两个亚基中的Gbetagamma结合位点不相同； GIRK1（而非 GIRK2）在 C 末端的前半部分具有先前未被识别的 Gbetagamma 相互作用片段。在两个亚基中发现的主要 C 端 Gbetagamma 结合片段大约位于氨基酸 320 和 409 之间（根据 GIRK1 计数）。 GIRK1 中 C 端亮氨酸 262 或 333 的突变（之前被认为对通道的 Gbetagamma 调节至关重要）以及 GIRK2 中相应亮氨酸 273 和 344 的突变显着改变了通过非洲爪蟾卵母细胞中表达的 GIRK1/GIRK2 通道的 K+ 电流的特性，但没有明显减少 Gbetagamma 与相应融合蛋白的结合，表明这些残基主要是对于调节 Gbetagamma 诱导的通道门控变化（而不是 Gbetagamma 结合）很重要。
G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gbetagamma. The binding sites of Gbetagamma in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gbetagamma has not been studied. We verified and extended the map of Gbetagamma-binding sites in GIRK1 by using two approaches: direct binding of Gbetagamma to fragments of GIRK subunits (pull down), and competition of these fragments with the Galpha(i1) subunit for binding to Gbetagamma. We also mapped the Gbetagamma-binding sites in GIRK2. In both subunits, the N terminus binds Gbetagamma. In the C terminus, the Gbetagamma-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gbetagamma-interacting segments in the first half of the C terminus. The main C-terminal Gbetagamma-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gbetagamma regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gbetagamma to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gbetagamma-induced changes in channel gating rather than Gbetagamma binding.