Mutant GFP- Based FRET Analysis of K+ Channel Organization
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The fact that genetic incorporation of the green fluorescent protein GFP (1) into an ion channel does not usually abolish function of either protein (see Part 2 of this book) enables a researcher to localize an ion channel in a living cell at almost every step of its biogenesis. Indeed, by means of light and confocal microscopy ion channels have been observed through the major steps of the secretory pathway: ER-Golgi- TGN- plasma membrane (2, 3, 4). Single GFP tagging can be used to sort out various trafficking mutants (5) and to demonstrate ion channel interaction with other proteins (3,6, 7, 8). Potentially this approach allows the monitoring of ion channel trafficking and turnover in real time. The advantage of such in vivo localization is partly compromised by the necessity of labeling and detecting a control protein specific for a given compartment-usually achieved by immunostaining followed by cell fixation. This complication was bypassed by the development of blue, cyan, yellow, red, and other mutant fluorescent proteins (BFP, CFP, YFP, and RFP, respectively), that make it possible to monitor two proteins labeled with optically distinguishable fluorescent tags almost simultaneously in vivo and localize one protein relative to another. Ion channel examples are absent for the moment, application for other proteins is reviewed in ref. 9. Such co-coloring allows attributing two proteins roughly to the same compartment but does not reveal their closer association owing to the limited spatial resolution of light microscopy.
KeywordsFluorescence Resonance Energy Transfer KATP Channel Fluorescence Resonance Energy Transfer Efficiency Fluorescence Resonance Energy Transfer Signal Fluorescence Resonance Energy Transfer Analysis
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