[Show abstract][Hide abstract] ABSTRACT: Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine's immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.
The Journal of Immunology 11/2013; 191(12). DOI:10.4049/jimmunol.1300702 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effector memory T cells (TM) play a key role in the pathology of certain autoimmune disorders. The activity of effector TM cells is under the control of Kv1.3 ion channels, which facilitate the Ca(2+) influx necessary for T cell activation and function, i.e. cytokine release and proliferation. Consequently, the knock-down of Kv1.3 expression in effector TM's may be utilized as a therapy for the treatment of autoimmune diseases. In this study we synthesized lipid unilamellar nanoparticles (NPs) that can selectively deliver Kv1.3 siRNAs into TM cells in vitro. NPs made from a mixture of phosphatidylcholine, pegylated/biotinylated phosphoethanolamine and cholesterol were functionalized with biotinylated-CD45RO (cell surface marker of TM's) antibodies via fluorophore-conjugated streptavidin (CD45RO-NPs). Incubation of T cells with CD45RO-NPs resulted into the selective attachment and endocytosis of the NPs into TM's. Furthermore, the siRNA against Kv1.3, encapsulated into the CD45RO-NPs, was released into the cytosol. Consequently, the expression of Kv1.3 channels decreased significantly in TM's, which led to a remarkable decrease in Ca(2+) influx. Our results can form the basis of an innovative therapeutic approach in autoimmunity.
[Show abstract][Hide abstract] ABSTRACT: The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca(2+) levels. Ca(2+) is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ∼6 µm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca(2+) levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.
PLoS ONE 08/2012; 7(8):e43859. DOI:10.1371/journal.pone.0043859 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The NH(2) terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH(2)-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH(2) terminus of NBCe1A are important in its accurate targeting to the plasma membrane.
[Show abstract][Hide abstract] ABSTRACT: The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. K(V)1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates K(V)1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating K(V)1.3 and the signaling pathway underneath. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited K(V)1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI(6-22). Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on K(V)1.3. Overexpression of a constitutively active mutant of Lck reduced the response of K(V)1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds K(V)1.3 to Lck, abolished PKA modulation of K(V)1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with K(V)1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates K(V)1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the K(V)1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.
[Show abstract][Hide abstract] ABSTRACT: The response of T cells to antigens (T cell activation) is marked by an increase in intracellular Ca²⁺ levels. Voltage-gated and Ca²⁺-dependent K⁺ channels control the membrane potential of human T cells and regulate Ca²⁺ influx. This regulation is dependent on proper accumulation of K⁺ channels at the immunological synapse (IS) a signaling zone that forms between a T cell and antigen presenting cell. It is believed that the IS provides a site for regulation of the activation response and that K⁺ channel inhibition occurs at the IS, but the underlying mechanisms are unknown. A mathematical model was developed to test whether K⁺ efflux through K⁺ channels leads to an accumulation of K⁺ in the IS cleft, ultimately reducing K⁺ channel function and intracellular Ca²⁺ concentration ([Ca²⁺](i)). Simulations were conducted in models of resting and activated T cell subsets, which express different levels of K⁺ channels, by varying the K⁺ diffusion constant and the spatial localization of K⁺ channels at the IS. K⁺ accumulation in the IS cleft was calculated to increase K⁺ concentration ([K⁺]) from its normal value of 5.0 mM to 5.2-10.0 mM. Including K⁺ accumulation in the model of the IS reduced calculated K⁺ current by 1-12% and consequently, reduced calculated [Ca²⁺](i) by 1-28%. Significant reductions in K⁺ current and [Ca²⁺](i) only occurred in activated T cell simulations when most K⁺ channels were centrally clustered at the IS. The results presented show that the localization of K⁺ channels at the IS can produce a rise in [K⁺] in the IS cleft and lead to a substantial decrease in K⁺ currents and [Ca²⁺](i) in activated T cells thus providing a feedback inhibitory mechanism during T cell activation.
[Show abstract][Hide abstract] ABSTRACT: Biomaterials, especially those based on nanomaterials, have emerged as critical tools in biomedical applications. The applications encompass a wide range such as implantable devices, tissue regeneration, drug delivery, diagnostic systems, and molecular printing. The type of materials used also covers a wide range: metals (permanent and degradable), polymers (permanent and degradable), carbon nanotubes, and lipid nanoparticles. This paper explores the use of microfluidic platforms as a high-throughput research tool for the evaluation of nanobiomaterials. Typical screening of such materials involves cell/tissue cultures to determine attributes such as cell adhesion, proliferation, differentiation, as well as biocompatibility. In addition to this, other areas such as drug delivery and toxicity can also be evaluated via microfluidics. Traditional approach for screening of such materials is very time-consuming, and a lot of animals should be sacrificed since it involves one material and a single composition or concentration for a single test. The microfluidics approach has the advantage of using multiple types of drugs and their concentration gradients to simultaneously study the effect on the nanobiomaterial and its interaction with cell/tissue. In addition to this, microfluidics provides a unique environment to study the effect of cell-to-extracellular interaction and cell-to-cell communication in the presence of the nanobiomaterials.
Journal of Nanomaterials 01/2012; 2012(1687-4110). DOI:10.1155/2012/789841 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxia in solid tumors contributes to decreased immunosurveillance via down-regulation of Kv1.3 channels in T lymphocytes and associated T cell function inhibition. However, the mechanisms responsible for Kv1.3 down-regulation are not understood. We hypothesized that chronic hypoxia reduces Kv1.3 surface expression via alterations in membrane trafficking. Chronic hypoxia decreased Kv1.3 surface expression and current density in Jurkat T cells. Inhibition of either protein synthesis or degradation and endocytosis did not prevent this effect. Instead, blockade of clathrin-coated vesicle formation and forward trafficking prevented the Kv1.3 surface expression decrease in hypoxia. Confocal microscopy revealed an increased retention of Kv1.3 in the trans-Golgi during hypoxia. Expression of adaptor protein-1 (AP1), responsible for clathrin-coated vesicle formation at the trans-Golgi, was selectively down-regulated by hypoxia. Furthermore, AP1 down-regulation increased Kv1.3 retention in the trans-Golgi and reduced Kv1.3 currents. Our results indicate that hypoxia disrupts AP1/clathrin-mediated forward trafficking of Kv1.3 from the trans-Golgi to the plasma membrane thus contributing to decreased Kv1.3 surface expression in T lymphocytes.
[Show abstract][Hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) T cells exhibit several activation signaling anomalies including defective Ca(2+) response and increased NF-AT nuclear translocation. The duration of the Ca(2+) signal is critical in the activation of specific transcription factors and a sustained Ca(2+) response activates NF-AT. Yet, the distribution of Ca(2+) responses in SLE T cells is not known. Furthermore, the mechanisms responsible for Ca(2+) alterations are not fully understood. Kv1.3 channels control Ca(2+) homeostasis in T cells. We reported a defect in Kv1.3 trafficking to the immunological synapse (IS) of SLE T cells that might contribute to the Ca(2+) defect. The present study compares single T cell quantitative Ca(2+) responses upon formation of the IS in SLE, normal, and rheumatoid arthritis (RA) donors. Also, we correlated cytosolic Ca(2+) concentrations and Kv1.3 trafficking in the IS by two-photon microscopy. We found that sustained [Ca(2+)](i) elevations constitute the predominant response to antigen stimulation of SLE T cells. This defect is selective to SLE as it was not observed in RA T cells. Further, we observed that in normal T cells termination of Ca(2+) influx is accompanied by Kv1.3 permanence in the IS, while Kv1.3 premature exit from the IS correlates with sustained Ca(2+) responses in SLE T cells. Thus, we propose that Kv1.3 trafficking abnormalities contribute to the altered distribution in Ca(2+) signaling in SLE T cells. Overall these defects may explain in part the T cell hyperactivity and dysfunction documented in SLE patients.
[Show abstract][Hide abstract] ABSTRACT: The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca(2+) response. Kv1.3 channels control Ca(2+) homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca(2+) signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca(2+) response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca(2+) response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca(2+) response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca(2+) response, and disruption of this process can account for alterations of downstream Ca(2+)-dependent signaling events.
The Journal of Immunology 11/2009; 183(10):6296-302. DOI:10.4049/jimmunol.0900613 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tiny medicine refers to the development of small easy to use devices that can help in the early diagnosis and treatment of disease. Early diagnosis is the key to successfully treating many diseases. Nanomaterial-based biosensors utilize the unique properties of biological and physical nanomaterials to recognize a target molecule and effect transduction of an electronic signal. In general, the advantages of nanomaterial-based biosensors are fast response, small size, high sensitivity, and portability compared to existing large electrodes and sensors. Systems integration is the core technology that enables tiny medicine. Integration of nanomaterials, microfluidics, automatic samplers, and transduction devices on a single chip provides many advantages for point of care devices such as biosensors. Biosensors are also being used as new analytical tools to study medicine. Thus this paper reviews how nanomaterials can be used to build biosensors and how these biosensors can help now and in the future to detect disease and monitor therapies.
[Show abstract][Hide abstract] ABSTRACT: Understanding the molecular mechanisms underlying dysregulated immune responses in human immunodeficiency virus type 1 (HIV-1) infection is crucial for the control of HIV/AIDS. Despite the postulate that HIV envelope glycoprotein gp120-CD4 interactions lead to impaired T-cell responses, the precise mechanisms underlying such association are not clear. To address this, we analyzed Lck and F-actin redistribution into the immunological synapse in stimulated human primary CD4(+) T cells from HIV-1-infected donors. Similar experiments were performed with CD4(+) T cells from HIV-uninfected donors, which were exposed to anti-CD4 domain 1 antibodies, as an in vitro model of gp120-CD4 interactions, or aldithriol-inactivated HIV-1 virions before stimulation. CD4(+) T cells from HIV-infected patients exhibited a two- to threefold inhibition of both Lck and F-actin recruitment into the synapse, compared to cells from uninfected donors. Interestingly, defective recruitment of Lck was ameliorated following suppressive highly active antiretroviral therapy. Engagement of the CD4 receptor on T cells from HIV-uninfected donors before anti-CD3/CD28 stimulation led to similar defects. Furthermore, the redistribution of Lck into lipid rafts was abrogated by CD4 preengagement. Our results suggest that the engagement of CD4 by HIV gp120 prior to T-cell receptor stimulation leads to dysregulation of early signaling events and could consequently play an important role in impaired CD4(+) T-cell function.
Journal of Virology 12/2008; 83(3):1193-200. DOI:10.1128/JVI.01023-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This article reviews the state of the art in carbon nanotube electrode and biosensor research. Carbon nanotubes have unique mechanical, electrical, and geometrical properties that are ideal for developing different types of nanoscale electrodes and biosensors. Carbon nanotube synthesis and subsequent functionalization strategies to immobilize special biomolecules are discussed first. Then different types of carbon nanotube biosensors and electroanalytical methods are reviewed particularly considering their capabilities for low detection limits, point-of-care applications, and label-free use. Detection strategies for proteins and nucleic acids, as well as mammalian and bacterial cells are also outlined. We conclude with some speculations and predictions on future exciting and challenging directions for nanotube biosensor research and applications.
[Show abstract][Hide abstract] ABSTRACT: Aberrant T cell responses during T cell activation and immunological synapse (IS) formation have been described in systemic lupus erythematosus (SLE). Kv1.3 potassium channels are expressed in T cells where they compartmentalize at the IS and play a key role in T cell activation by modulating Ca(2+) influx. Although Kv1.3 channels have such an important role in T cell function, their potential involvement in the etiology and progression of SLE remains unknown. This study compares the K channel phenotype and the dynamics of Kv1.3 compartmentalization in the IS of normal and SLE human T cells. IS formation was induced by 1-30 min exposure to either anti-CD3/CD28 Ab-coated beads or EBV-infected B cells. We found that although the level of Kv1.3 channel expression and their activity in SLE T cells is similar to normal resting T cells, the kinetics of Kv1.3 compartmentalization in the IS are markedly different. In healthy resting T cells, Kv1.3 channels are progressively recruited and maintained in the IS for at least 30 min from synapse formation. In contrast, SLE, but not rheumatoid arthritis, T cells show faster kinetics with maximum Kv1.3 recruitment at 1 min and movement out of the IS by 15 min after activation. These kinetics resemble preactivated healthy T cells, but the K channel phenotype of SLE T cells is identical to resting T cells, where Kv1.3 constitutes the dominant K conductance. The defective temporal and spatial Kv1.3 distribution that we observed may contribute to the abnormal functions of SLE T cells.
The Journal of Immunology 08/2007; 179(1):346-56. DOI:10.4049/jimmunol.179.1.346 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Na(+):HCO(3)(-) cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins.
American journal of physiology. Renal physiology 05/2007; 292(4):F1245-55. DOI:10.1152/ajprenal.00410.2006 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca(2+) influx. KCa3.1 channels modulate Ca(2+) signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3epsilon, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3epsilon to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15-30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca(2+) influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.