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.
