Finley Melissa, Arrabit Christine, Fowler Catherine, Suen Ka Fai, Slesinger Paul A
Peptide Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
J Physiol. 2004 Mar 16;555(Pt 3):643-57. doi: 10.1113/jphysiol.2003.056101. Epub 2004 Jan 14.
The activity of G protein-activated inwardly rectifying K(+) channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G(betagamma) subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying G(betagamma) activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G(betagamma) binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2(L344E) and GIRK2(G347H), that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of G(betagamma) subunits. Combining the two mutations (GIRK2(EH)) led to a more severe reduction in carbachol-activated and G(betagamma)-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2(L344E) and GIRK2(EH) also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G(betagamma)-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G(betagamma) activation. In vitro G(betagamma) binding assays revealed an approximately 60% decrease in G(betagamma) binding to the C-terminal domain of GIRK2(L344E) but no statistical change with GIRK2(EH) or GIRK2(G347H), though both mutants exhibited G(betagamma)-impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G(betagamma) activation of GIRK2 channels. Based on the 1.8 A structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the betaL-betaM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G(betagamma) alters the interaction between the betaL-betaM loop and the N-terminal domain.
G蛋白激活的内向整流钾通道（GIRK或Kir3）的活性对于调节神经元、心脏和内分泌细胞的膜兴奋性很重要。尽管已知Gβγ亚基可结合GIRK通道的N端和C端，但Gβγ激活GIRK的潜在机制尚不清楚。在这里，我们使用由GIRK2和IRK1（一种对G蛋白不敏感的内向整流器）构建的嵌合体和点突变体，来确定GIRK2中对Gβγ结合和激活重要的区域。对非洲爪蟾卵母细胞中表达的突变通道的分析揭示了GIRK2 C端结构域中的两个氨基酸取代，即GIRK2（L344E）和GIRK2（G347H），它们在共表达Gβγ亚基时表现出卡巴胆碱激活电流降低，但基础电流显著增强。将这两个突变结合（GIRK2（EH））导致卡巴胆碱激活电流和Gβγ刺激电流更严重的降低。然而，乙醇激活电流正常，这表明不依赖G蛋白的门控不受这些突变的影响。当在HEK - 293T细胞中表达时，GIRK2（L344E）和GIRK2（EH）也显示出卡巴胆碱激活降低和乙醇激活正常。使用在HEK - 293T细胞中表达的表位标记通道，免疫细胞化学显示Gβγ功能受损的突变体在质膜上表达，尽管程度不同，并且不能完全解释Gβγ激活的降低。体外Gβγ结合试验显示Gβγ与GIRK2（L344E）C端结构域的结合减少了约60%，但GIRK2（EH）或GIRK2（G347H）没有统计学变化，尽管这两个突变体都表现出Gβγ激活受损。总之，这些结果表明L344以及在较小程度上的G347在Gβγ激活GIRK2通道中起重要的功能作用。基于GIRK1胞质结构域的1.8 Å结构，L344和G347位于βL - βM环中，该环远离孔道且靠近N端结构域。根据一个激活模型对结果进行了讨论，在该模型中Gβγ改变了βL - βM环与N端结构域之间的相互作用。
