A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes

Christine Beeton, Heike Wulff, Satendra Singh, Steve Botsko, George Crossley, George A. Gutman, Michael D. Cahalan, Michael Pennington, K. George Chandy

Research output: Contribution to journalArticle

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Abstract

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 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 Kv1.3high expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.

Original languageEnglish (US)
Pages (from-to)9928-9937
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number11
DOIs
StatePublished - Mar 14 2003

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T-cells
Up-Regulation
T-Lymphocytes
Flow cytometry
Antigens
Rats
Myelin Sheath
Flow Cytometry
Protein Kinase C
Animals
Chemical activation
Autoimmune Experimental Encephalomyelitis
ShK-F6CA
Tissue
Calcium
Multiple Sclerosis
Limit of Detection
Electric potential
Animal Models
Staining and Labeling

ASJC Scopus subject areas

  • Biochemistry

Cite this

A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes. / Beeton, Christine; Wulff, Heike; Singh, Satendra; Botsko, Steve; Crossley, George; Gutman, George A.; Cahalan, Michael D.; Pennington, Michael; Chandy, K. George.

In: Journal of Biological Chemistry, Vol. 278, No. 11, 14.03.2003, p. 9928-9937.

Research output: Contribution to journalArticle

Beeton, C, Wulff, H, Singh, S, Botsko, S, Crossley, G, Gutman, GA, Cahalan, MD, Pennington, M & Chandy, KG 2003, 'A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes', Journal of Biological Chemistry, vol. 278, no. 11, pp. 9928-9937. https://doi.org/10.1074/jbc.M212868200
Beeton, Christine ; Wulff, Heike ; Singh, Satendra ; Botsko, Steve ; Crossley, George ; Gutman, George A. ; Cahalan, Michael D. ; Pennington, Michael ; Chandy, K. George. / A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 11. pp. 9928-9937.
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AU - Crossley, George

AU - Gutman, George A.

AU - Cahalan, Michael D.

AU - Pennington, Michael

AU - Chandy, K. George

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AB - 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 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 Kv1.3high expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.

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