Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes ...mellitus or rheumatoid arthritis (RA) are mainly CD4⁺CCR7⁻CD45RA⁻ effector memory T cells ($T_{EM}$cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naíve or central-memory ($T_{CM}$) cells. In$T_{EM}$cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP,$p56^{Ick}$, and CD4. Although Kv1.3 inhibitors ShK(L5)-amide (SL5) and PAP1 do not prevent immunological synapse formation, they suppress Ca²⁺-signaling, cytokine production, and proliferation of autoantigen-specific$T_{EM}$cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.
The voltage-gated Kv1.3 K(+) channel is a novel target for immunomodulation of autoreactive effector memory T (T(EM)) cells that play a major role in the pathogenesis of autoimmune diseases. We ...describe the characterization of the novel peptide ShK(L5) that contains l-phosphotyrosine linked via a nine-atom hydrophilic linker to the N terminus of the ShK peptide from the sea anemone Stichodactyla helianthus. ShK(L5) is a highly specific Kv1.3 blocker that exhibits 100-fold selectivity for Kv1.3 (K(d) = 69 pM) over Kv1.1 and greater than 250-fold selectivity over all other channels tested. ShK(L5) suppresses the proliferation of human and rat T(EM) cells and inhibits interleukin-2 production at picomolar concentrations. Naive and central memory human T cells are initially 60-fold less sensitive than T(EM) cells to ShK(L5) and then become resistant to the peptide during activation by up-regulating the calcium-activated K(Ca)3.1 channel. ShK(L5) does not exhibit in vitro cytotoxicity on mammalian cell lines and is negative in the Ames test. It is stable in plasma and when administered once daily by subcutaneous injection (10 mug/kg) attains "steady state" blood levels of approximately 300 pM. This regimen does not cause cardiac toxicity assessed by continuous EKG monitoring and does not alter clinical chemistry and hematological parameters after 2-week therapy. ShK(L5) prevents and treats experimental autoimmune encephalomyelitis and suppresses delayed type hypersensitivity in rats. ShK(L5) might prove useful for therapy of autoimmune disorders.
The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ...ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org.
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
The voltage-gated Kv1.3 channel and the Ca
2+-activated IKCa1 K
+ channel are expressed in T cells in a distinct pattern that depends on the state of lymphocyte activation and differentiation. The ...channel phenotype changes during the progression from the resting to the activated cell state and from naı̈ve to effector memory cells, affording promise for specific immunomodulatory actions of K
+ channel blockers. In this article, we review the functional roles of these channels in both naı̈ve cells and memory cells, describe the development of selective inhibitors of Kv1.3 and IKCa1 channels, and provide a rationale for the potential therapeutic use of these inhibitors in immunological disorders.
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 â¼600
channels per cell. Rat and human T cells that had been repeatedly stimulated 7â10 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 1â3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific
rat T cell line following myelin antigen stimulation, peaking at 15â20 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.
Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K
...+-channel-blocking peptides belonging to family 2 peptides and comprising 30–40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named α-KTx1–12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.
PDF
