title: Generation of functional fluorescent BK channels by random insertion of GFP variants.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA.
authors.forename: Teresa
authors.initials: T
authors.lastname: Giraldez
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Hughes
authors.forename: Fred J
authors.initials: FJ
authors.lastname: Sigworth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D006224:Cricetinae; D004251:DNA Transposable Elements / Q000235:genetics / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology
chemical_list: D004251:DNA Transposable Elements; D051036:Large-Conductance Calcium-Activated Potassium Channels; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium