Gyorgy Panyi

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

K+ channel blockers: Novel tools to inhibit T cell activation leading to specific immunosuppression

During the last two decades since the identification and characterization of T cell potassium channels great advances have been made in the understanding of the role of these channels in T cell functions, especially in antigen-induced activation. Their limited tissue distribution and the recent discovery that different T cell subtypes carrying out distinct immune functions show specific expression levels of these channels have made T cell potassium channels attractive targets for immunomodulatory drugs. Many toxins of various animal species and a structurally diverse array of small molecules inhibiting these channels with varying affinity and selectivity were found and their successful use in immunosuppression in vivo was also demonstrated. Better understanding of the topological differences between potassium channel pores, detailed knowledge of toxin and small-molecule structures and the identification of the binding sites of blocking compounds make it possible to improve the selectivity and affinity of the lead compounds by introducing modifications based on structural information. In this review the basic properties and physiological roles of the voltage-gated Kv1.3 and the Ca2+-activated IKCa1 potassium channels are discussed along with an overview of compounds inhibiting these channels and approaches aiming at producing more efficient modulators of immune functions for the treatment of diseases like sclerosis multiplex and type I diabetes.

Two novel toxins from the Amazonian scorpion Tityus cambridgei that block Kv1.3 and Shaker B K+-channels with distinctly different affinities

Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.

Cross Talk between Activation and Slow Inactivation Gates of Shaker Potassium Channels

This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs+ ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.

Vm24, a natural immunosuppressive peptide, potently and selectively blocks Kv1.3 potassium channels of human T cells.

Blockade of Kv1.3 K+ channels in T cells is a promising therapeutic approach for the treatment of autoimmune diseases such as multiple sclerosis and type 1 diabetes mellitus. Vm24 (α-KTx 23.1) is a novel 36-residue Kv1.3-specific peptide isolated from the venom of the scorpion Vaejovis mexicanus smithi. Vm24 inhibits Kv1.3 channels of human lymphocytes with high affinity (Kd = 2.9 pM) and exhibits >1500-fold selectivity over other ion channels assayed. It inhibits the proliferation and Ca2+ signaling of human T cells in vitro and reduces delayed-type hypersensitivity reactions in rats in vivo. Our results indicate that Vm24 has exceptional pharmacological properties that make it an excellent candidate for treatment of certain autoimmune diseases.

Biophysical and pharmacological aspects of K+ channels in T lymphocytes.

Voltage-gated Kv1.3 and Ca -activated IKCa1 K channels play a pivotal role in antigendependent activation and proliferation of lymphocytes. These channels primarily determine the membrane potential of T cells, and thus regulate the magnitude of the Ca signal required for efficient gene transcription and subsequent proliferation. Although these facts are generally well described and acknowledged, some recent discoveries have motivated research in this field, which is reviewed herein along with the basic biophysical characterization of Kv1.3 and IKCa1. The discovery of T cell subset-specific expression of Kv1.3 points towards the potential therapeutic use of high affinity and high specificity Kv1.3 inhibitors as specific immunosuppressors in the management of autoimmune diseases, such as Multiple Sclerosis. In meeting the demands for an ideal immunosuppressor, several laboratories have discovered potent natural Kv1.3specific inhibitors and engineered peptides which have a better pharmacological profile based on the biophysical characterization of the interaction surface between the channel pore and the toxins. In contrast to the generally accepted permissive role of Kv1.3 during lymphocyte activation, the discovery of the localization of Kv1.3 in the immunological synapse might open new opportunities in the regulation of T cell activation by this channel species. ___________________________________________ Abbreviations [Ca]i: cytosolic free Ca 2 + concentration; CaM: calmodulin; ChTx: charybdotoxin; CLSM: confocal laser scanning microscopy; CRAC: calcium-release activated ___________________________________________ György Panyi University of Debrecen, Medical and Health Science Center, Research Center for Molecular Medicine, Department of Biophysics and Cell Biology 98. Nagyerdei krt. Debrecen, 4012 Hungary email:panyi@jaguar.dote.hu Ca channel; CTLs: cytotoxic T cells; FRET: fluorescence resonance energy transfer; IP3: 1,4,5-inositol trisphosphate; IS: immunological synapse; KcsA: prokaryotic K channel from Streptomyces lividans;; Kv1.3/FLAG: FLAG epitopetagged Kv1.3; KvAP: prokaryotic voltage-dependent K channel from Aeropyrum pernix; PKC: protein kinase C; ShK: Stichodactyla helianthus peptide; TCR/CD3: T cell receptor/CD3 complex; TCM: central memory T cell; TEM: effector memory T cell ___________________________________________ Overview of lymphocyte activation, role of ion channels The generation of an efficient immune response requires the clonal expansion of lymphocytes recognizing a given antigen specifically. Antigens are presented to the T cells by professional antigen presenting cells; both antigen presentation and recognition are mediated by membrane proteins. Major histocompatibility complex proteins (MHC) of antigen presenting cells loaded with processed antigens are sampled by the T cell receptor/CD3 complex (TCR/CD3) of T cells. Specific interaction between the presented antigen and the TCR/CD3 complex activates a variety of transmembrane signaling pathways involving non-receptor tyrosine kinases. The initial events depend primarily on the activation of two Srcfamily kinases Lck and Fyn. Phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMS) allows further kinases to be recruited and activated by different phosphorylation cascades. Some of the protein kinase pathways, such as the Ras/MAP pathway, contribute directly to the regulation of gene transcription required for the proliferation of T cells. The other consequence of the specifically increased tyrosine kinase activity is the activation of phospholipase C-γ (PLCγ) (Fig. 1). This enzyme cleaves a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, to yield diacylglycerol and 1,4,5Neue Datei AV.indd Abs1:515 06.09.2005 10:16:41

Ion channels in T lymphocytes: An update on facts, mechanisms and therapeutic targeting in autoimmune diseases

During the last quarter of a century a large body of evidence was gathered about the involvement of ion channels in T lymphocyte activation. A series of remarkable findings promoted T cell ion channels to become potential pharmaceutical targets in the therapy of autoimmune disorders. Numerous comprehensive reviews describe the types of ion channels found in the plasma membrane of T cells and their roles in signaling pathways leading to activation, the changes in the expression of these channels brought upon by differentiation to various T cell subsets, the formation and possible functions of signaling molecular clusters that include ion channels in the immunological synapse, the discovery and refinement of structurally different ion channel blockers and the successful in vivo application of such compounds to suppress hypersensitivity reactions and autoimmune processes. In this review we wish to provide a concise update on these topics from recent years, highlighting the most notable developments.

Voltage-Gated Sodium Channel Nav1.7 Maintains the Membrane Potential and Regulates the Activation and Chemokine-Induced Migration of a Monocyte-Derived Dendritic Cell Subset

Expression of CD1a protein defines a human dendritic cell (DC) subset with unique functional activities. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a− and CD1a+ DC. Single-cell electrophysiology (patch-clamp) and quantitative PCR experiments performed on sorted CD1a− and CD1a+ immature DC (IDC) showed that the frequency of cells expressing Na+ current, current density, and the relative expression of the SCN9A gene encoding Nav1.7 were significantly higher in CD1a+ cells than in their CD1a− counterparts. The activity of Nav1.7 results in a depolarized resting membrane potential (−8.7 ± 1.5 mV) in CD1a+ IDC as compared with CD1a− cells lacking Nav1.7 (−47 ± 6.2 mV). Stimulation of DC by inflammatory signals or by increased intracellular Ca2+ levels resulted in reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a+ IDC, resulting in decreased cell migration, whereas pharmacological inhibition of Nav1.7 by tetrodotoxin sensitized the cells for activation signals. Fine-tuning of IDC functions by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and cytokine responses of these DC subsets.

Developmental Switch of the Expression of Ion Channels in Human Dendritic Cells

Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca2+ signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca2+-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.

Structure, function, and chemical synthesis of Vaejovis mexicanus peptide 24: a novel potent blocker of Kv1.3 potassium channels of human T lymphocytes.

Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) ∼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/β motif consisting of a single-turn α-helix and a three-stranded antiparallel β-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.