DETECTING CONFORMATIONAL CHANGES IN GIRK CHANNELS

离子通道的结构信息远远落后于可溶性蛋白质的结构信息。同样，关于离子通道在激活过程中发生的重要构象变化的信息也很少。在这里，我建议开发一种新的基于荧光的方法来测量分子内距离，以及6蛋白门控K+通道（GIRKs）激活过程中的构象变化。该方法依赖于附着在通道上的两个荧光团之间的荧光共振能量转移。我将使用增强型青色荧光蛋白（ECFP）作为供体，荧光素衍生物染料FlAsH作为受体。FlAsH特异性结合α -螺旋四胱氨酸基序C-C-X-X-C-C (Cys4)，该基序可以放置在通道的不同部分。通过测量ECFP和FlAsH之间的FRET，我可以获得体内通道不同区域相对距离的详细信息。我将使用这种技术来检查在不同生理刺激（包括g蛋白β - γ亚基）激活时，GIRK通道的构象变化。我还将尝试通过检测FlAsH由于环境变化而导致的荧光特性的变化来监测通道中的构象变化。在初步实验中，我将Cys4基序和ECFP连接到GIRK4通道的c端。通道是功能性的，FlAsH/ECFP荧光定位于质膜。我的提案中的实验将提供有关在门控过程中GIRK通道动态构象变化的信息。

Structural information about ion channels lags far behind that of soluble proteins. Similarly little information is available about functionally important conformational changes ion channels undergo during activation. Here I propose to develop a new fluorescence based approach to measure intramolecular distances, as well as conformational changes during activation in 6-protein gated K+ channels (GIRKs). The method relies on fluorescence resonance energy transfer (FRET) between two fluorophores attached to the channel. I will use the enhanced cyan fluorescent protein (ECFP) as donor and a fluorescein derivative dye, FlAsH, as acceptor. FlAsH binds specifically to an alpha helical tetracysteine motif C-C-X-X-C-C (Cys4) that can be placed in different parts of the channel. By measuring FRET between ECFP and FlAsH, I can obtain detailed information on the relative distances of different regions of the channel in vivo. I will use this technique to examine conformational changes in GIRK channels upon activation by different physiological stimuli including the G-protein beta-gamma subunits. I will also attempt to monitor conformational changes in the channel by detecting changes in the fluorescent properties of FlAsH due to changes in its environment. In preliminary experiments, I attached the Cys4 motif as well as ECFP to the C-terminus of GIRK4 channels. The channels were functional and FlAsH/ECFP fluorescence localized to the plasma membrane. The experiments in my proposal will provide information about dynamic conformation changes in GIRK channels during gating.