Antioxidant Carbon Nanoparticles Inhibit Fibroblast-Like Synoviocyte Invasiveness and Reduce Disease Severity in a Rat Model of Rheumatoid Arthritis.

Reactive oxygen species have been involved in the pathogenesis of rheumatoid arthritis (RA). Our goal was to determine the effects of selectively scavenging superoxide (O2•-) and hydroxyl radicals with antioxidant nanoparticles, called poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), on the pathogenic functions of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and on the progression of an animal model of RA. We used human FLS from patients with RA to determine PEG-HCC internalization and effects on FLS cytotoxicity, invasiveness, proliferation, and production of proteases. We used the pristane-induced arthritis (PIA) rat model of RA to assess the benefits of PEG-HCCs on reducing disease severity. PEG-HCCs were internalized by RA-FLS, reduced their intracellular O2•-, and reduced multiple measures of their pathogenicity in vitro, including proliferation and invasion. In PIA, PEG-HCCs caused a 65% reduction in disease severity, as measured by a standardized scoring system of paw inflammation and caused a significant reduction in bone and tissue damage, and circulating rheumatoid factor. PEG-HCCs did not induce lymphopenia during PIA. Our study demonstrated a role for O2•- and hydroxyl radicals in the pathogenesis of a rat model of RA and showed efficacy of PEG-HCCs in treating a rat model of RA.

Inhibition of Upf2-Dependent Nonsense-Mediated Decay Leads to Behavioral and Neurophysiological Abnormalities by Activating the Immune Response.

In humans, disruption of nonsense-mediated decay (NMD) has been associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability. However, the mechanism by which deficient NMD leads to neurodevelopmental dysfunction remains unknown, preventing development of targeted therapies. Here we identified novel protein-coding UPF2 (UP-Frameshift 2) variants in humans with NDD, including speech and language deficits. In parallel, we found that mice lacking Upf2 in the forebrain (Upf2 fb-KO mice) show impaired NMD, memory deficits, abnormal long-term potentiation (LTP), and social and communication deficits. Surprisingly, Upf2 fb-KO mice exhibit elevated expression of immune genes and brain inflammation. More importantly, treatment with two FDA-approved anti-inflammatory drugs reduced brain inflammation, restored LTP and long-term memory, and reversed social and communication deficits. Collectively, our findings indicate that impaired UPF2-dependent NMD leads to neurodevelopmental dysfunction and suggest that anti-inflammatory agents may prove effective for treatment of disorders with impaired NMD.

Targeting KCa1.1 Channels with a Scorpion Venom Peptide for the Therapy of Rat Models of Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLSs) are a key cell type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLSs and found that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects. In this study, we aimed to determine whether the KCa1.1 ß1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch-clamp and functional assays were used to determine whether IbTX can regulate FLSs through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined whether IbTX causes side effects including incontinence or tremors in rats, compared with those treated with the small-molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 were expressed by FLSs in joints of rats with experimental arthritis. IbTX inhibited KCa1.1 channels expressed by FLSs from patients with RA and by FLSs from rat models of RA and reduced FLS invasiveness. IbTX significantly reduced disease severity in two rat models of RA. Unlike paxilline, IbTX did not induce tremors or incontinence in rats. Overall, IbTX inhibited KCa1.1 channels on FLSs and treated rat models of RA without inducing side effects associated with nonspecific KCa1.1 blockade and could become the basis for the development of a new treatment of RA.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog.

Effector memory T lymphocytes (TEM cells) that lack expression of CCR7 are major drivers of inflammation in a number of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. The Kv1.3 potassium channel is a key regulator of CCR7- TEM cell activation. Blocking Kv1.3 inhibits TEM cell activation and attenuates inflammation in autoimmunity, and as such, Kv1.3 has emerged as a promising target for the treatment of TEM cell-mediated autoimmune diseases. The scorpion venom-derived peptide HsTX1 and its analog HsTX1[R14A] are potent Kv1.3 blockers and HsTX1[R14A] is selective for Kv1.3 over closely-related Kv1 channels. PEGylation of HsTX1[R14A] to create a Kv1.3 blocker with a long circulating half-life reduced its affinity but not its selectivity for Kv1.3, dramatically reduced its adsorption to inert surfaces, and enhanced its circulating half-life in rats. PEG-HsTX1[R14A] is equipotent to HsTX1[R14A] in preferential inhibition of human and rat CCR7- TEM cell proliferation, leaving CCR7+ naïve and central memory T cells able to proliferate. It reduced inflammation in an active delayed-type hypersensitivity model and in the pristane-induced arthritis (PIA) model of rheumatoid arthritis (RA). Importantly, a single subcutaneous dose of PEG-HsTX1[R14A] reduced inflammation in PIA for a longer period of time than the non-PEGylated HsTX1[R14A]. Together, these data indicate that HsTX1[R14A] and PEG-HsTX1[R14A] are effective in a model of RA and are therefore potential therapeutics for TEM cell-mediated autoimmune diseases. PEG-HsTX1[R14A] has the additional advantages of reduced non-specific adsorption to inert surfaces and enhanced circulating half-life.

Changes in Gene Expression and Metabolism in the Testes of the Rat following Spinal Cord Injury.

Spinal cord injury (SCI) results in devastating changes to almost all aspects of a patient's life. In addition to a permanent loss of sensory and motor function, males also will frequently exhibit a profound loss of fertility through poorly understood mechanisms. We demonstrate that SCI causes measureable pathology in the testis both acutely (24 h) and chronically up to 1.5 years post-injury, leading to loss in sperm motility and viability. SCI has been shown in humans and rats to induce leukocytospermia, with the presence of inflammatory cytokines, anti-sperm antibodies, and reactive oxygen species found within the ejaculate. Using messenger RNA and metabolomic assessments, we describe molecular and cellular changes that occur within the testis of adult rats over an acute to chronic time period. From 24 h, 72 h, 28 days, and 90 days post-SCI, the testis reveal a distinct time course of pathological events. The testis show an acute drop in normal sexual organ processes, including testosterone production, and establishment of a pro-inflammatory environment. This is followed by a subacute initiation of an innate immune response and loss of cell cycle regulation, possibly due to apoptosis within the seminiferous tubules. At 1.5 years post-SCI, there is a chronic low level immune response as evidenced by an elevation in T cells. These data suggest that SCI elicits a wide range of pathological processes within the testes, the actions of which are not restricted to the acute phase of injury but rather extend chronically, potentially through the lifetime of the subject. The multiplicity of these pathological events suggest a single therapeutic intervention is unlikely to be successful.

Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts.

Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1.

Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation.

Autoimmune diseases mediated by a type of white blood cell-T lymphocytes-are currently treated using mainly broad-spectrum immunosuppressants that can lead to adverse side effects. Antioxidants represent an alternative approach for therapy of autoimmune disorders; however, dietary antioxidants are insufficient to play this role. Antioxidant carbon nanoparticles scavenge reactive oxygen species (ROS) with higher efficacy than dietary and endogenous antioxidants. Furthermore, the affinity of carbon nanoparticles for specific cell types represents an emerging tactic for cell-targeted therapy. Here, we report that nontoxic poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), known scavengers of the ROS superoxide (O2•-) and hydroxyl radical, are preferentially internalized by T lymphocytes over other splenic immune cells. We use this selectivity to inhibit T cell activation without affecting major functions of macrophages, antigen-presenting cells that are crucial for T cell activation. We also demonstrate the in vivo effectiveness of PEG-HCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity and in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Our results suggest the preferential targeting of PEG-HCCs to T lymphocytes as a novel approach for T lymphocyte immunomodulation in autoimmune diseases without affecting other immune cells.

Characterization of a novel MR-detectable nanoantioxidant that mitigates the recall immune response.

In many human diseases, the presence of inflammation is associated with an increase in the level of reactive oxygen species (ROS). The resulting state of oxidative stress is highly detrimental and can initiate a cascade of events that ultimately lead to cell death. Thus, many therapeutic attempts have been focused on either modulating the immune system to lower inflammation or reducing the damaging caused by ROS. Berlin et al. reported the development of a novel nanoantioxidant known as poly(ethylene glycol)-functionalized-hydrophilic carbon clusters (PEG-HCCs). They showed that PEG-HCCs could be targeted to cancer cells, utilized as a drug delivery vector, and can even be visualized ex vivo. Our work here furthers this work and characterizes Gd-DTPA conjugated PEG-HCCs and explores the potential for in vivo tracking of T cells in live mice. We utilized a mouse model of delayed-type hypersensitivity (DTH) to assess the immunomodulatory effects of PEG-HCCs. The T1 -agent Gd-DTPA was then conjugated to the PEG-HCCs and T1 measurements, and T1 -weighted MRI of the modified PEG-HCCs was done to assess their relaxivity. We then assessed if PEG-HCCs could be visualized both ex vivo and in vivo within the mouse lymph node and spleen. Mice treated with PEG-HCCs showed significant improvements in the DTH assay as compared to the vehicle (saline)-treated control. Flow cytometry demonstrated that splenic T cells are capable of internalizing PEG-HCCs whereas fluorescent immunohistochemistry showed that PEG-HCCs are detectable within the cortex of lymph nodes. Finally, our nanoantioxidants can be visualized in vivo within the lymph nodes and spleen of a mouse after addition of the Gd-DTPA. PEG-HCCs are internalized by T cells in the spleen and can reduce inflammation by suppression of a recall immune response. PEG-HCCs can be modified to allow for both in vitro and in vivo visualization using MRI. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Different expression of ß subunits of the KCa1.1 channel by invasive and non-invasive human fibroblast-like synoviocytes.

BACKGROUND: Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA-FLS) contribute to joint inflammation and damage characteristic of the disease. RA-FLS express KCa1.1 (BK, Slo1, MaxiK, KCNMA1) as their major plasma membrane potassium channel. Blocking KCa1.1 reduces the invasive phenotype of RA-FLS and attenuates disease severity in animal models of RA. This channel has therefore emerged as a promising therapeutic target in RA. However, the pore-forming α subunit of KCa1.1 is widely distributed in the body, and blocking it induces severe side effects, thus limiting its value as a therapeutic target. On the other hand, KCa1.1 channels can also contain different accessory subunits with restricted tissue distribution that regulate channel kinetics and pharmacology. Identification of the regulatory subunits of KCa1.1 expressed by RA-FLS may therefore provide the opportunity for generating a selective target for RA treatment. METHODS: Highly invasive RA-FLS were isolated from patients with RA, and FLS from patients with osteoarthritis (OA) were used as minimally invasive controls. The ß subunit expression by FLS was assessed by quantitative reverse transcription polymerase chain reactions, Western blotting, and patch-clamp electrophysiology combined with pharmacological agents. FLS were sorted by flow cytometry on the basis of their CD44 expression level for comparison of their invasiveness and with their expression of KCa1.1 α and ß subunits. ß1 and ß3 subunit expression was reduced with small interfering RNA (siRNA) to assess their specific role in KCa1.1α expression and function and in FLS invasiveness. RESULTS: We identified functional ß1 and ß3b regulatory subunits in RA-FLS. KCa1.1 ß3b subunits were expressed by 70 % of the cells and were associated with highly invasive CD44(high) RA-FLS, whereas minimally invasive CD44(low) RA-FLS and OA-FLS expressed either ß1 subunit. Furthermore, we found that silencing the ß3 but not the ß1 subunit with siRNA reduced KCa1.1 channel density at the plasma membrane of RA-FLS and inhibited RA-FLS invasiveness. CONCLUSIONS: Our findings suggest the KCa1.1 channel composed of α and ß3b subunits as an attractive target for the therapy of RA.

N-Terminally extended analogues of the K⁺ channel toxin from Stichodactyla helianthus as potent and selective blockers of the voltage-gated potassium channel Kv1.3.

The voltage-gated potassium channel Kv1.3 is an important target for the treatment of autoimmune diseases and asthma. Blockade of Kv1.3 by the sea anemone peptide K⁺-channel toxin from Stichodactyla helianthus (ShK) inhibits the proliferation of effector memory T lymphocytes and ameliorates autoimmune diseases in animal models. However, the lack of selectivity of ShK for Kv1.3 over the Kv1.1 subtype has driven a search for Kv1.3-selective analogues. In the present study, we describe N-terminally extended analogues of ShK that contain a negatively-charged Glu, designed to mimic the phosphonate adduct in earlier Kv1.3-selective analogues, and consist entirely of common protein amino acids. Molecular dynamics simulations indicated that a Trp residue at position [-3] of the tetrapeptide extension could form stable interactions with Pro377 of Kv1.3 and best discriminates between Kv1.3 and Kv1.1. This led to the development of ShK with an N-terminal Glu-Trp-Ser-Ser extension ([EWSS]ShK), which inhibits Kv1.3 with an IC50 of 34 pm and is 158-fold selective for Kv1.3 over Kv1.1. In addition, [EWSS]ShK is more than 2900-fold more selective for Kv1.3 over Kv1.2 and KCa3.1 channels. As a highly Kv1.3-selective analogue of ShK based entirely on protein amino acids, which can be produced by recombinant expression, this peptide is a valuable addition to the complement of therapeutic candidates for the treatment of autoimmune diseases.

Development of highly selective Kv1.3-blocking peptides based on the sea anemone peptide ShK.

ShK, from the sea anemone Stichodactyla helianthus, is a 35-residue disulfide-rich peptide that blocks the voltage-gated potassium channel Kv1.3 at ca. 10 pM and the related channel Kv1.1 at ca. 16 pM. We developed an analog of this peptide, ShK-186, which is currently in Phase 1b-2a clinical trials for the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. While ShK-186 displays a >100-fold improvement in selectivity for Kv1.3 over Kv1.1 compared with ShK, there is considerable interest in developing peptides with an even greater selectivity ratio. In this report, we describe several variants of ShK that incorporate p-phophono-phenylalanine at the N-terminus coupled with internal substitutions at Gln16 and Met21. In addition, we also explored the combinatorial effects of these internal substitutions with an alanine extension at the C-terminus. Their selectivity was determined by patch-clamp electrophysiology on Kv1.3 and Kv1.1 channels stably expressed in mouse fibroblasts. The peptides with an alanine extension blocked Kv1.3 at low pM concentrations and exhibited up to 2250-fold selectivity for Kv1.3 over Kv1.1. Analogs that incorporates p-phosphono-phenylalanine at the N-terminus blocked Kv1.3 with IC50s in the low pM range and did not affect Kv1.1 at concentrations up to 100 nM, displaying a selectivity enhancement of >10,000-fold for Kv1.3 over Kv1.1. Other potentially important Kv channels such as Kv1.4 and Kv1.6 were only partially blocked at 100 nM concentrations of each of the ShK analogs.

KCa1.1 inhibition attenuates fibroblast-like synoviocyte invasiveness and ameliorates disease in rat models of rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) participate in joint inflammation and damage in rheumatoid arthritis (RA) and its animal models. The purpose of this study was to define the importance of KCa1.1 (BK, Maxi-K, Slo1, KCNMA1) channel expression and function in FLS and to establish these channels as potential new targets for RA therapy. METHODS: We compared KCa1.1 expression levels in FLS from rats with pristane-induced arthritis (PIA) and in FLS from healthy rats. We then used ex vivo functional assays combined with small interfering RNA-induced knockdown, overexpression, and functional modulation of KCa1.1 in PIA FLS. Finally, we determined the effectiveness of modulating KCa1.1 in 2 rat models of RA, moderate PIA and severe collagen-induced arthritis (CIA). RESULTS: We found that PIA FLS expressed the KCa1.1 channel as their major potassium channel, as has been found in FLS from patients with RA. In contrast, FLS from healthy rats expressed fewer of these channels. Inhibiting the function or expression of KCa1.1 ex vivo reduced proliferation and invasive properties of, as well as protease production by, PIA FLS, whereas opening native KCa1.1 or overexpressing the channel enhanced the invasiveness of both FLS from rats with PIA and FLS from healthy rats. Treatment with a KCa1.1 channel blocker at the onset of clinical signs stopped disease progression in the PIA and CIA models, reduced joint and bone damage, and inhibited FLS invasiveness and proliferation. CONCLUSION: Our results demonstrate a critical role of KCa1.1 channels in the regulation of FLS invasiveness and suggest that KCa1.1 channels represent potential therapeutic targets in RA.

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

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.

Blocking KV1.3 channels inhibits Th2 lymphocyte function and treats a rat model of asthma.

Allergic asthma is a chronic inflammatory disease of the airways. Of the different lower airway-infiltrating immune cells that participate in asthma, T lymphocytes that produce Th2 cytokines play important roles in pathogenesis. These T cells are mainly fully differentiated CCR7(-) effector memory T (TEM) cells. Targeting TEM cells without affecting CCR7(+) naïve and central memory (TCM) cells has the potential of treating TEM-mediated diseases, such as asthma, without inducing generalized immunosuppression. The voltage-gated KV1.3 potassium channel is a target for preferential inhibition of TEM cells. Here, we investigated the effects of ShK-186, a selective KV1.3 channel blocker, for the treatment of asthma. A significant proportion of T lymphocytes in the lower airways of subjects with asthma expressed high levels of KV1.3 channels. ShK-186 inhibited the allergen-induced activation of peripheral blood T cells from those subjects. Immunization of F344 rats against ovalbumin followed by intranasal challenges with ovalbumin induced airway hyper-reactivity, which was reduced by the administration of ShK-186. ShK-186 also reduced total immune infiltrates in the bronchoalveolar lavage and number of infiltrating lymphocytes, eosinophils, and neutrophils assessed by differential counts. Rats with the ovalbumin-induced model of asthma had elevated levels of the Th2 cytokines IL-4, IL-5, and IL-13 measured by ELISA in their bronchoalveolar lavage fluids. ShK-186 administration reduced levels of IL-4 and IL-5 and induced an increase in the production of IL-10. Finally, ShK-186 inhibited the proliferation of lung-infiltrating ovalbumin-specific T cells. Our results suggest that KV1.3 channels represent effective targets for the treatment of allergic asthma.

A potent and Kv1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases.

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels Kv1.3 and Kv1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2 kcal/mol gain for the Kv1.3/Kv1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for Kv1.3 in the low picomolar range and a selectivity of more than 2,000-fold for Kv1.3 over Kv1.1. This remarkable potency and selectivity for Kv1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.

A potent and selective peptide blocker of the Kv1.3 channel: prediction from free-energy simulations and experimental confirmation.

The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.

Blocking KCa3.1 channels increases tumor cell killing by a subpopulation of human natural killer lymphocytes.

Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy.

Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers.

The K(v)1.3 voltage-gated potassium channel regulates membrane potential and calcium signaling in human effector memory T cells that are key mediators of autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Thus, subtype-specific K(v)1.3 blockers have potential for treatment of autoimmune diseases. Several K(v)1.3 channel blockers have been characterized from scorpion venom, all of which have an α/ß scaffold stabilized by 3-4 intramolecular disulfide bridges. Chemical synthesis is commonly used for producing these disulfide-rich peptides but this approach is time consuming and not cost effective for production of mutants, fusion proteins, fluorescently tagged toxins, or isotopically labelled peptides for NMR studies. Recombinant production of K(v)1.3 blockers in the cytoplasm of E. coli generally necessitates oxidative refolding of the peptides in order to form their native disulfide architecture. An alternative approach that avoids the need for refolding is expression of peptides in the periplasm of E. coli but this often produces low yields. Thus, we developed an efficient Pichia pastoris expression system for production of K(v)1.3 blockers using margatoxin (MgTx) and agitoxin-2 (AgTx2) as prototypic examples. The Pichia system enabled these toxins to be obtained in high yield (12-18 mg/L). NMR experiments revealed that the recombinant toxins adopt their native fold without the need for refolding, and electrophysiological recordings demonstrated that they are almost equipotent with the native toxins in blocking K(V)1.3 (IC(50) values of 201±39 pM and 97 ± 3 pM for recombinant AgTx2 and MgTx, respectively). Furthermore, both recombinant toxins inhibited T-lymphocyte proliferation. A MgTx mutant in which the key pharmacophore residue K28 was mutated to alanine was ineffective at blocking K(V)1.3 and it failed to inhibit T-lymphocyte proliferation. Thus, the approach described here provides an efficient method of producing toxin mutants with a view to engineering K(v)1.3 blockers with therapeutic potential.