G-protein alpha subunits in regulation of GIRK channels

描述（由申请人提供）：G蛋白激活的K+通道（GIRK; Kir 3）在心脏和大脑功能中发挥重要作用，介导许多神经递质的抑制作用。GIRK通道通过直接结合G蛋白的G-β和G-α亚基而被激活，也直接与GIRK相互作用，并在决定信号传导的特异性方面发挥重要作用。在过去的几年里，我们已经提出了证据表明，α亚基直接和间接影响GIRK门控。然而，许多现象学，生理影响，和GIRK的相互作用的Galalpha的机制仍然不清楚。这些问题是拟议研究的重点。我们的长期目标是了解GIRK的分子机制和Galpha亚基调节的生理意义，并利用GIRK来研究G蛋白通路中的特异性和G蛋白效应器相互作用的一般问题。具体目标是： (1)进一步研究Ga在GIRK门控中的作用和机制。通过在体外监测蛋白质-蛋白质相互作用，并通过使用在非洲爪蟾卵母细胞和HEK细胞中的生理测定、诱变、免疫细胞化学和荧光能量转移（FRET），我们将：i）验证GcdGIRK相互作用的存在及其对GIRK活性的影响; ii）阐明哪种形式的Gm在生理上是重要的; iii）绘制Galpha和GIRK之间相互作用的位点，并研究这些位点在通道门控和调节中的作用; iv）研究GIRK门控如何受Galpha影响 (2)了解Galpha-GIRK相互作用在确定该途径中信号传导特异性中的作用。将监测GIRK通道和各种Ga α亚基之间的物理相互作用，并将其与这些Ga α亚基的共表达对GIRK通道功能的影响相关联。 （三）、Ga对GIRK调节的生理相关性研究。GIRK调制的作用，在培养的心房肌细胞和神经元将使用电生理学方法，通过蛋白质的共表达，除了纯化的蛋白质和肽切除的补丁，并通过敲低内源性G蛋白亚基来解决。我们将利用生化和免疫组化方法探讨Galpha和GIRK在心房和脑中的共定位和复合物的形成。

DESCRIPTION (provided by applicant): G-protein activated K+ channels (GIRK; Kir3) play an important role in the cardiac and brain function, mediating the inhibitory action of many neurotransmitters. GIRK channels are activated by direct binding G-Beta and G-Alpha subunits of G-proteins also directly interact with GIRK and play an important role in determining the specificity of signaling. Over the last years, we have presented evidence that Galpha subunits affect GIRK gating directly and indirectly. However, much of the phenomenology, the physiological impact, and the mechanisms of interaction of Galpha with GIRK remains unclear. These issues are in the focus of the proposed study. Our long-term goal is to understand the molecular mechanisms and the physiological significance of GIRK modulation by Galpha subunits, and to utilize GIRK to study the general problem of specificity and G protein effector interactions in G protein pathways. The specific aims are: (1) Further study of the roles and mechanisms of actions of Ga in GIRK gating. By monitoring protein-protein interactions in vitro and by using physiological assays in Xenopus oocytes and HEK cells, mutagenesis, immunocytochemistry, and fluorescence energy transfer (FRET), we will: i) verify the existence of GcdGIRK interactions and their impact on GIRK activity; ii) clarify which form(s) of Gm is physiologically important; iii) map the sites of interaction between Galpha and GIRK, and study the role of these sites in channel gating and regulation; iv) investigate how GIRK gating is affected by Galpha (2) Understanding of the role of Galpha-GIRK interactions in determining specificity of signaling in this pathway. The physical interactions between the GIRK channel and various Galpha subunits will be monitored, and correlated with the effects of coexpression of these Galpha subunits, on the function of GIRK channels. (3). Study of the physiological correlates of modulation of GIRK by Ga. The roles of GIRK modulation by Galpha in cultured atrial myocytes and neurons will be addressed using electrophysiological methods, by co-expression of proteins, addition of purified proteins and peptides to excised patches, and by knock-down of endogenous G protein subunits. Colocalization and complex formation between Galpha and GIRK will be explored in atrium and brain using biochemical and immunohistochemical methods.