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J Biol Chem 2003 Jan 2
Beeton C, Wulff H, Singh S, Botsko S, Crossley G, Gutman GA, Cahalan
MD, Pennington M, Chandy KG.
Physiology and Biophysics, University of California, Irvine, Irvine,
CA 92697-4560.
T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3high 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 Kv1.3high 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 KV channels.
In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of ~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 hours and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity.
Both calcium- and PKC-dependent pathways were required for the antigen-induced Kv1.3 up-regulation.
ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3high expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.