A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes.
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.
Beeton
Christine
C
Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Wulff
Heike
H
Singh
Satendra
S
Botsko
Steve
S
Crossley
George
G
Gutman
George A
GA
Cahalan
Michael D
MD
Pennington
Michael
M
Chandy
K George
KG
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
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