title: A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes.
abstract: T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Beeton
authors.forename: Heike
authors.initials: H
authors.lastname: Wulff
authors.forename: Satendra
authors.initials: S
authors.lastname: Singh
authors.forename: Steve
authors.initials: S
authors.lastname: Botsko
authors.forename: George
authors.initials: G
authors.lastname: Crossley
authors.forename: George A
authors.initials: GA
authors.lastname: Gutman
authors.forename: Michael D
authors.initials: MD
authors.lastname: Cahalan
authors.forename: Michael
authors.initials: M
authors.lastname: Pennington
authors.forename: K George
authors.initials: KG
authors.lastname: Chandy
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000941:Antigens / Q000096:biosynthesis; D001710:Biotin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003064:Cnidarian Venoms / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D051664:Kv1.3 Potassium Channel; D008214:Lymphocytes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D015221:Potassium Channels / Q000096:biosynthesis* / Q000737:chemistry; D024642:Potassium Channels, Voltage-Gated; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution; D015854:Up-Regulation
chemical_list: D000596:Amino Acids; D000941:Antigens; D003064:Cnidarian Venoms; D005452:Fluoresceins; D005456:Fluorescent Dyes; C496973:KCNA3 protein, human; C496974:Kcna3 protein, mouse; C496975:Kcna3 protein, rat; D051664:Kv1.3 Potassium Channel; D010455:Peptides; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C095391:ShK neurotoxin; C473776:ShK-F6CA; D001710:Biotin; D011493:Protein Kinase C; D002118:Calcium