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Biomedical subjects

George A Gutman

Publications and source records attributed to George A Gutman.

12 recordsLinked to original sources
PubMed2006

Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases.

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 naïve or central-memory (T(CM)) cells. In T(EM) cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2, SAP97, ZIP, p56(lck), 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.

Animals↗
PubMed2006

The sense of place in the immune system.

This series of reviews examines the effect of differing tissue environments on the activity and functional capacity of cells in the immune system. From their origins as hematopoietic stem cells, throughout their development and as mature cells, cells of the immune system find themselves in distinct and highly specialized niches, and contact with antigen or inflammatory signals changes their phenotype, activity and trafficking. Two-photon microscopy has provided the first direct observations of living cells and their activation choreography in the tissue environment and will no doubt continue to provide greater understanding of cellular dynamics and immune function.

Animals↗
PubMed2005

Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases.

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.

Animals↗
PubMed2004

Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA.

The approximately 1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream approximately 1.0 kb and the downstream approximately 0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most approximately 0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream approximately 1.0 kb. Although the systematic deletion of sequences within the 3'-most approximately 0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.

5' Flanking Region↗
PubMed2004

K+ channels as targets for specific immunomodulation.

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 naïve 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 naïve 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.

Adjuvants, Immunologic↗
PubMed2004

Enhanced neuronal excitability in the absence of neurodegeneration induces cerebellar ataxia.

Cerebellar ataxia, a devastating neurological disease, may be initiated by hyperexcitability of deep cerebellar nuclei (DCN) secondary to loss of inhibitory input from Purkinje neurons that frequently degenerate in this disease. This mechanism predicts that intrinsic DCN hyperexcitability would cause ataxia in the absence of upstream Purkinje degeneration. We report the generation of a transgenic (Tg) model that supports this mechanism of disease initiation. Small-conductance calcium-activated potassium (SK) channels, regulators of firing frequency, were silenced in the CNS of Tg mice with the dominant-inhibitory construct SK3-1B-GFP. Transgene expression was restricted to the DCN within the cerebellum and was detectable beginning on postnatal day 10, concomitant with the onset of cerebellar ataxia. Neurodegeneration was not evident up to the sixth month of age. Recordings from Tg DCN neurons revealed loss of the apamin-sensitive after-hyperpolarization current (IAHP) and increased spontaneous firing through SK channel suppression, indicative of DCN hyperexcitability. Spike duration and other electrogenic conductance were unaffected. Thus, a purely electrical alteration is sufficient to cause cerebellar ataxia, and SK openers such as the neuroprotective agent riluzole may reduce neuronal hyperexcitability and have therapeutic value. This dominant-inhibitory strategy may help define the in vivo role of SK channels in other neuronal pathways.

Animals↗
PubMed2003

SK3-1C, a dominant-negative suppressor of SKCa and IKCa channels.

Small conductance Ca2+-activated K+ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BKCa or KV channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca2+-dependent inactivation of CaV channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K+ channels. SK3-1C may therefore suppress calmodulin-gated SKCa/IKCa channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.

Alternative Splicing↗
PubMed2003

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.

Amino Acids↗
PubMed2003

International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Phylogeny↗