The Journal of General Physiology
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Published online Oct 31 2005. doi:10.1085/jgp.200509368
The Rockefeller University Press, 0022-1295 $8.00
JGP, Volume 126, Number 5, 429-438
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ARTICLE

 Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants

Teresa Giraldez1, Thomas E. Hughes3, and Fred J. Sigworth1,2

1 Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520
2 Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT 06520
3 Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717

Correspondence to Fred J. Sigworth: fred.sigworth{at}yale.edu

The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the {alpha} subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The "success rate" of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.

Abbreviations used in this paper: CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; YFP, yellow fluorescent protein.


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