title: Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression.
abstract: The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells.
authors: Department of Biological Sciences, ‡Department of Chemistry, §Molecular Biosensor and Imaging Center, and #Center for the Neural Basis of Cognition, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Christopher P
authors: CP
authors: Pratt
authors: Department of Biological Sciences, ‡Department of Chemistry, §Molecular Biosensor and Imaging Center, and #Center for the Neural Basis of Cognition, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Jianjun
authors: J
authors: He
authors: Department of Biological Sciences, ‡Department of Chemistry, §Molecular Biosensor and Imaging Center, and #Center for the Neural Basis of Cognition, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Yi
authors: Y
authors: Wang
authors: Department of Biological Sciences, ‡Department of Chemistry, §Molecular Biosensor and Imaging Center, and #Center for the Neural Basis of Cognition, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Alison L
authors: AL
authors: Barth
authors: Department of Biological Sciences, ‡Department of Chemistry, §Molecular Biosensor and Imaging Center, and #Center for the Neural Basis of Cognition, Carnegie Mellon University , 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Marcel P
authors: MP
authors: Bruchez
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D003116:Color; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D010455:Peptides; D005576:Colforsin; D000262:Adenylyl Cyclases