DETECTING CONFORMATIONAL CHANGES IN GIRK CHANNELS

Project Details

Description

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.
StatusFinished
Effective start/end date3/1/012/28/02

Funding

  • National Institute of General Medical Sciences: $45,560.00

ASJC

  • Physiology

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