title: Random insertion of split-cans of the fluorescent protein venus into Shaker channels yields voltage sensitive probes with improved membrane localization in mammalian cells.
abstract: FlaSh-YFP, a fluorescent protein (FP) voltage sensor that is a fusion of the Shaker potassium channel with yellow fluorescent protein (YFP), is primarily expressed in the endoplasmic reticulum (ER) of mammalian cells, possibly due to misfolded monomers. In an effort to improve plasma membrane expression, the FP was split into two non-fluorescent halves. Each half was randomly inserted into Shaker monomers via a transposon reaction. Shaker subunits containing the 5' half were co-expressed with Shaker subunits containing the 3' half. Tetramerization of Shaker subunits is required for re-conjugation of the FP. The misfolded monomers trapped in ER are unlikely to tetramerize and reconstitute the beta-can structure, and thus intracellular fluorescence might be reduced. This split-can transposon approach yielded 56 fluorescent probes, 30 (54%) of which were expressed at the plasma membrane and were capable of optically reporting changes in membrane potential. The largest signal from these novel FP-sensors was a -1.4% in ΔF/F for a 100 mV depolarization, with on time constants of about 15 ms and off time constants of about 200 ms. This split-can transposon approach has the potential to improve other multimeric probes.
authors.affiliation: Department of Physiology, Yale University, New Haven, CT, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Jin
authors.forename: Bradley
authors.initials: B
authors.lastname: Baker
authors.forename: Robbie
authors.initials: R
authors.lastname: Mealer
authors.forename: Lawrence
authors.initials: L
authors.lastname: Cohen
authors.forename: Vincent
authors.initials: V
authors.lastname: Pieribone
authors.forename: Arnd
authors.initials: A
authors.lastname: Pralle
authors.forename: Thomas
authors.initials: T
authors.lastname: Hughes
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D003600:Cytosol / Q000737:chemistry / Q000648:ultrastructure; D004251:DNA Transposable Elements; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D020125:Mutation, Missense; D009447:Neuroblastoma / Q000473:pathology; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D051637:Shaker Superfamily of Potassium Channels; C054562:yellow fluorescent protein, Bacteria