George Chandy, M.D., Ph.D. - US grants

membrane channels; nucleic acid probes; lymphocyte;

Potassium (K) channels control a wide array of physiological processes, including regulation of membrane potential and cell volume in diverse tissues, and modulation of electrical excitability in the nervous system. Our studies have revealed three distinct types of K channels in T lymphocytes (n, n' and l) that play a role in mitogen-activation and differentiation. Expression of these channels is dependent on the phenotype and on the activation and differentiation status of T cells. We have also identified a link between altered K channel expression in T lymphocytes and four disparate autoimmune disorders: systemic lupus erythematosus, type-1 diabetes mellitus, rheumatoid arthritis and multiple sclerosis. Using molecular techniques we have characterized the genes encoding the types n and l K channels in T cells; the human chromosomal locations of 14 related K channel genes have been determined. Anti-peptide antibodies against the T cell K channel proteins have allowed us to start measuring the rate of turnover of channel proteins in T cells. In this proposal we plan to extend our studies to further define the role of K channels in T cell activation and differentiation. The overall goal of the proposed experiments is three-fold. First, we will map the transcripts encoding T cell K channels, and identify tissue-specific and activation-dependent regulatory elements. Second, we will measure the rate of synthesis and degradation of the T cell K channel transcripts during T and B cell activation and development. Third, using anti-peptide antibodies we will study channel protein turnover in lymphoid cells. Comparison of normal and diseased T cells may reveal abnormalities In channel-gene or protein regulation associated with autoimmunity. In the course of these studies we may reveal DNA binding proteins that modulate K channel-gene activity during T cell proliferation.

Potassium (K) channels control a wide array of physiological processes, including regulation of membrane potential and cell volume in diverse tissues, and modulation of electrical excitability in the nervous system. Our studies have revealed three distinct types of K channels in T lymphocytes (n, n' and l) that play a role in mitogen-activation and differentiation. Expression of these channels is dependent on the phenotype and on the activation and differentiation status of T cells. We have also identified a link between altered K channel expression in T lymphocytes and four disparate autoimmune disorders: systemic lupus erythematosus, type-1 diabetes mellitus, rheumatoid arthritis and multiple sclerosis. Using molecular techniques we have characterized the genes encoding the types n and l K channels in T cells; the human chromosomal locations of 14 related K channel genes have been determined. Anti-peptide antibodies against the T cell K channel proteins have allowed us to start measuring the rate of turnover of channel proteins in T cells. In this proposal we plan to extend our studies to further define the role of K channels in T cell activation and differentiation. The overall goal of the proposed experiments is three-fold. First, we will map the transcripts encoding T cell K channels, and identify tissue-specific and activation-dependent regulatory elements. Second, we will measure the rate of synthesis and degradation of the T cell K channel transcripts during T and B cell activation and development. Third, using anti-peptide antibodies we will study channel protein turnover in lymphoid cells. Comparison of normal and diseased T cells may reveal abnormalities In channel-gene or protein regulation associated with autoimmunity. In the course of these studies we may reveal DNA binding proteins that modulate K channel-gene activity during T cell proliferation.

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Voltage-gated K (Kv) channels regulate the behavior of diverse cell types. An extended family of 19 genes encode these important proteins. Delineation of the tertiary structure of any one of these channels will provide the conceptual framework for understanding ion permeation, activation, inactivation and deactivation, and be the basis for developing models of related ion channels. Such structural information could also guide the rational design of novel ion channel modulating drugs that could be used for the therapy for a wide range of conditions including diabetes, cardiac arrhythmias, stroke, autoimmune disorders, hypertension, urinary incontinence and male pattern baldness. We propose to perform a detailed structural analysis of Kv1.3, a critical modulator of T-lymphocyte function. The Kv 1.3 channel is one of the best characterized Kv channels and hundreds of micrograms of tetrameric, functional purified Kv1.3 protein are now available for structural studies. Combining complementary site-specific mutagenesis, electrophysiology, computer modeling, protein chemistry and electron crystallography into two complementary strategies, we will probe the structure of Kv1.3. The first will extend our use of scorpion toxins as molecular calipers to identify new toxin:channel interactions and refine our model of the Kv1.3 pore and surrounding regions. Using this model as a guide, we will determine Kv1.3's interactions with a new class of high- affinity organic blockers of Kv1.3, which could then serve as a template for the design of more selective and potent blockers of the channel. Second, we will generate coherent two-dimensional Kv1.3 crystals in lipid membranes and determine the structure of Kv1.3 using electron crystallography. A density map will be generated on the basis of electron and optical diffraction data collected at multiple tilt angles. The Kv1.3 polypeptide will be fitted to this map, the toxin-mapping information being valuable in its interpretation. Our goal will be to reconstruct a three-dimensional image of the channel from the 2D information.

Several human hereditary neurological diseases are caused by expanded CAG repeats although the pathophysiological mechanism remains uncertain. Longer CAG alleles have also been reported to be over-represented in patients with bipolar disorder and schizophrenia, but the defective gene(s) remain unidentified. We isolated a human gene encoding a calcium-activated potassium channel (hSKCa3), found in neurons and containing a novel CAG repeat. Encoding a polyglutamine repeat near the amino terminus, this CAG repeat is highly polymorphic in normals, and long alleles are over-represented in schizophrenia and bipolar disorder. We now propose to combine the strengths of the four principal investigators in molecular biology, human genetics, neuroanatomy, biochemistry, and electrophysiology, to develop a detailed biophysical and pharmacological "fingerprint" of this channel. Using a chimeric strategy between hKCa3 and its "repeat-free" relative, hKCa4, coupled with site-specific mutagenesis, we will define functional domains within these proteins, and determine the effects of longer polyglutamine repeats on channel function. In-situ hybridization studies on human brains, from controls and patients with schizophrenia, will ascertain the neuronal distribution of hKCa3 in relation to neurotransmitter receptors and other channels, and potentially pinpoint key anatomical areas that might be implicated in schizophrenia. By defining the intron/exon organization of this gene, we will be able to initiate screening studies to identify point mutations in hKCa3 that might be associated with schizophrenia. We will also define the precise location of this gene with respect to other genetic markers for schizophrenia. The identification of the native promoter for hKCa3 will set the stage for the generation of transgenic mice that over-express hKCa3 with long polyglutamine repeats in the relevant regions of the brain; such mice might exhibit altered behavior. These experiments provide the framework for understanding the role of polyglutamine repeats in a protein of known function. The long term goal of these studies is to understand the role of hKCa3 in the pathogenesis of schizophrenia, and to develop specific modulators of this channel for potential use in the therapy of this debilitating neuropsychiatric disorder.

DESCRIPTION (provided by applicant): Functional blockade or elimination of antigen-specific immune responses without impacting general immune function must be considered a holy grail in the quest to treat autoimmune disease. One approach to achieving this lofty goal is to suppress and/or eliminate effector memory (TEM) cells that have been implicated in the pathogenesis of many autoimmune diseases without affecting other immune cells. This proposal focuses on the voltage-gated Kv1.3 potassium channel in TEM cells as a therapeutic target for autoimmune diseases. Disease-associated autoreactive T cells in patients with multiple sclerosis (MS), type-1 diabetes mellitus (T1DM) and rheumatoid arthritis (RA) are TEM cells with elevated expression of Kv1.3 channels. We have developed highly specific inhibitors of Kv1.3 and these selectively suppress calcium signaling, cytokine production, proliferation, and in vivo migration of TEM cells without affecting other T cell subsets. In proof-of-concept studies, Kv1.3 blockers prevent and/or treat disease in rat models of MS, T1DM, RA, contact dermatitis, delayed type hypersensitivity (DTH) and bone resorption associated with periodontitis. These blockers have excellent safety profiles in animal models. They do not compromise the acute protective immune response to viral and bacterial pathogens. Specific Kv1.3 blockers, provide an exciting new therapeutic approach to mute autoreactive responses without compromising the protective immune response. The studies outlined in this proposal will lay the groundwork for clinical trials in patients with MS with ShK-186, our lead candidate. Aim 1 will define the mechanisms underlying ShK-186's therapeutic effect in chronic relapsing-remitting experimental autoimmune encephalomyelitis (CR-EAE) in DA rats, a model for human MS. We will test the hypothesis that ShK-186 eliminates disease-causing CNS autoantigen-specific TEM cells by a mechanism we term death by neglect while other immune cells, particularly disease-suppressing regulatory T cells, proliferate unabated because they are protected by the ShK-186-resistant KCa3.1 channel. Aim 2 will characterize the therapeutic dose, frequency and duration of ShK-186 treatment in CR-EAE to better predict human studies. In Aim 3 we will use patch-clamp recording and flow cytometry to determine whether Kv1.3high expression is a reliable biomarker for MS, and whether it will be useful for tracking Kv1.3 blocker therapy. A second goal of aim 3 is to study channel expression in T cells from Cynomolgus monkeys because this species is widely used for toxicological evaluation of human immune modulating therapies and is our non-human primate choice for IND- enabling toxicological studies. We will define the K-channel phenotype of cynomolgus monkey T cell subsets, and we will evaluate ShK-186's effectiveness in suppressing monkey TEM cell-proliferation. PUBLIC HEALTH RELEVANCE: Our goal is to develop a therapy that suppresses or eliminates disease-causing immune cells in autoimmune diseases without compromising the protective immune response. Our strategy is to selectively target voltage-gated Kv1.3 potassium channels in effector memory T cells that are important mediators of autoimmune diseases.

[unreadable] DESCRIPTION (provided by applicant): Autoreactive memory T-lymphocytes are implicated in the pathogenesis of autoimmune diseases. Safe therapies that target these cells without generalized immunosuppression would have immense medical value. Using single-cell patch-clamp analysis in conjunction with confocal microscopy and flow cytometry, we have demonstrated that disease-associated T cells from patients with type-1 diabetes mellitus (T1DM), rheumatoid arthritis (RA) and multiple sclerosis (MS) express elevated levels of the Kv1.3 potassium channel and are CCR7-CD45RA- effector memory (TEM) T cells. We have developed highly specific Kv1.3 channel inhibitors and these inhibitors preferentially suppress cytokine production and proliferation of autoantigen-specific Kv1.3(high) TEM cells while sparing other T cells. Moreover, Kv1.3 inhibitors reduce the incidence of spontaneously developing experimental autoimmune DM in BB-WOR rats by limiting beta-cell destruction and they ameliorate disease in rat models of MS and RA without associated toxicity. In this exploratory R21 grant, we will test the hypothesis that the "Kv1.3highTEM phenotype" is a functional biomarker of disease-associated autoreactive T cells and will prove useful to monitor disease progression in people at high risk and also track autoimmune responses during therapy. In AIM-1 we will examine whether CD4+ T cells specific for GAD65 (274-286), IGRP (247-258) or insulin (A1-15) and pentamer-sorted CD8+ T cells specific for insulin (B10-18), pplAPP (5-13) and a novel epitope of IGRP (152-160) from patients with new-onset T1DM exhibit the Kv1,3highCCR7-CD45RA- TEM pattern. In AIM-2, multimers that give the most consistent results will be used to screen blood samples from patients with new onset T1DM (<1 year post diagnosis), established diabetes (>5 year duration), individuals at-risk for developing T1DM (anti-islet autoantibody positive) and healthy control subjects for the presence of multimer+ Kv1.3high TEM cells. Markers that are found to be predictive or informative in disease progression will be used (in subsequent studies) to assess blood samples obtained from HLA-DRB1-0401-positive individuals participating in ongoing clinical trials with immunotherapeutic interventions. In AIM-3 we will test whether Kv1.3 inhibitors suppress cytokine production and proliferatiion of high- and low-avidity autoantigen-specific multimer+ cells with equal effectiveness. This experiment has important implications if Kv1.3-specific blockers are to be developed as therapeutics. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PET/CT Scan Method to Monitor Pancreatic Beta-Cell Loss in Diabetes Mellitus