George K. Chandy

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases

The voltage‐gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK‐186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)‐related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51‐residue peptide expressed in the anterior secretory glands of the dog‐infecting hookworm Ancylostoma caninum and the human‐infecting hookworm Ancylostoma ceylanicum, and BmK1, the C‐terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar‐micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7‐ effector memory T cells without affecting naive and central memory subsets and inhibit the delayed‐type hypersensitivity (DTH) response caused by skin‐homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK‐related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.—Chhabra, S., Chang, S. C., Nguyen, H. M., Huq, R., Tanner, M. R., Londono, L. M., Estrada, R., Dhawan, V., Chauhan, S., Upadhyay, S. K., Gindin, M., Hotez, P. J., Valenzuela, J. G., Mohanty, B., Swarbrick, J. D., Wulff, H., Iadonato, S. P., Gutman, G. A., Beeton, C., Pennington, M. W., Norton, R. S., Chandy, K. G. Kv1.3 channel‐blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases. FASEB J. 28, 3952‐3964 (2014). www.fasebj.org

Enrichment of NK cells from human blood with the RosetteSep kit from StemCell technologies.

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differentiation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (>80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Transcription of the T-Cell Potassium Channel Kv1.3 Is Regulated by a GC-Rich TATA-Less Promoter

The basal promoter of Kv1.3, the voltage-gated K+ channel in T cells, lies within a GC-rich SmaI/SacI fragment lacking a TATA box and contains two SP1 sites together with two repeats of the motif GGGCGG. The promoters of many housekeeping genes as well as three related potassium channel genes (Kv1.4, Kv1.5, and Kv3.1) are also TATA-less and GC-rich, suggesting that all these genes may use common transcriptional regulation mechanisms. Upstream positive regulatory (enhancer) elements lie in a region containing two overlapping TFΠD sites and two SP1 motifs. Primer extension shows that the 5-NCR of Kv1.3 is significantly shorter (length 150 bp) than the corresponding regions ( > 1,000 bp) of other closely related genes (Kv1.1, Kv1.2, Kv1.4). The 5-NCR and the entire coding region of Kv1.3 are contained in a single uninterrupted exon spanning ∼1.65 kb. The diversity of Kv1.3 mRNAs (3.5-9.5 kb) found in lymphocytes and brain may, therefore, result from either alternate splicing in the 3-UTR or alternate use of polyadenylation sites.

Structural and Biochemical Features of the Kv1.3 Potassium Channel: An Aid to Guided Drug Design

The Kv1.3 potassium channel in T lymphocytes plays a major role in mitogen-induced activation and is widely recognized as a potential therapeutic target for immunosuppressive agents. Availability of s

Fluorinated General Anesthetics Modulate Shaker Potassium Channels at Clinically Relevant Concentrations

Recent studies demonstrated that selective intra-thalamic block of the Kv1.2 potassium channels rapidly reverses the unconsciousness of inhalational anesthesia (Alkire et al., 2009). Furthermore, intra-thalamic block of Kv1.3 with selective blocker, ShK, reverses the unconsciousness effect of sevoflurane. Both findings suggestthat Shaker-related potassium channels might be key targets for anesthetics. We tested this hypothesis using patch clamp recordings of potassium currents in cell lines stably expressing homomeric human Shaker family channels. We found that at low voltages, sub-clinical and clinical doses of sevoflurane irreversibly potentiate potassium current through Kv1.3>Kv1.2>Kv1.5>Kv1.1. channels, by increasing the peak current amplitude and accelerating the current activation kinetics. At higher voltages, clinical doses of sevoflurane inhibit Kv1.4>Kv1.5.>Kv1.3 and potentiate Kv1.2>Kv1.1. Sevoflurane had no effect on lipid bilayer conductance, suggesting direct interaction of the anesthetic with potassium channel subunits. Thus, Shaker potassium channel modulation may contribute to the clinical effects of general anesthesia. Supported by the Hillblom Foundation and NIH 1P01AG032131.