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Large-conductance calcium-activated potassium channel (KCa1.1; BK, Slo1, MaxiK, KCNMA1) is the predominant potassium channel expressed at the plasma membrane of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) isolated from the synovium of patients with RA. It is a critical regulator of RA-FLS migration and invasion and therefore represents an attractive target for the therapy of RA. However, the molecular mechanisms by which KCa1.1 regulates RA-FLS invasiveness have remained largely unknown. Here, we demonstrate that KCa1.1 regulates RA-FLS adhesion through controlling the plasma membrane expression and activation of β1 integrins, but not α4, α5, or α6 integrins. Blocking KCa1.1 disturbs calcium homeostasis, leading to the sustained phosphorylation of Akt and the recruitment of talin to β1 integrins. Interestingly, the pore-forming α subunit of KCa1.1 coimmunoprecipitates with β1 integrins, suggesting that this physical association underlies the functional interaction between these molecules. Together, these data outline a new signaling mechanism by which KCa1.1 regulates β1-integrin function and therefore invasiveness of RA-FLSs.-Tanner, M. R., Pennington, M. W., Laragione, T., Gulko, P. S., Beeton, C. KCa1.1 channels regulate β1-integrin function and cell adhesion in rheumatoid arthritis fibroblast-like synoviocytes.

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+ naive 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.

Matrix metalloproteinases (MMPs) represent more than 20 zinc-containing endopeptidases that cleave internal peptide bonds, leading to protein degradation. They play a critical role in many physiological cell functions, including tissue remodeling, embryogenesis, and angiogenesis. They are also involved in the pathogenesis of a vast array of diseases, including but not limited to systemic inflammation, various cancers, and cardiovascular, neurological, and autoimmune diseases. Here, we describe gel zymography to detect MMPs in cell and tissue samples and in cell culture supernatants.

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.Highly invasive RA-FLS were isolated from patients with RA, and FLS from patients with osteoarthritis (OA) were used as minimally invasive controls. The β 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 β subunits. β1 and β3 subunit expression was reduced with small interfering RNA (siRNA) to assess their specific role in KCa1.1α expression and function and in FLS invasiveness.We identified functional β1 and β3b regulatory subunits in RA-FLS. KCa1.1 β3b 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 β1 subunit. Furthermore, we found that silencing the β3 but not the β1 subunit with siRNA reduced KCa1.1 channel density at the plasma membrane of RA-FLS and inhibited RA-FLS invasiveness.Our findings suggest the KCa1.1 channel composed of α and β3b subunits as an attractive target for the therapy of RA.

The outcome of a malignant disease depends on the efficacy of the immune system to destroy cancer cells. Key steps in this process, for example the generation of a proper Ca 2+ signal induced by recognition of a specific antigen, are regulated by various ion channel including voltage-gated Kv1.3 and Ca 2+ -activated KCa3.1 K + channels, and the interplay between Orai and STIM to produce the Ca 2+ -release-activated Ca 2+ (CRAC) current required for T-cell proliferation and function. Understanding the immune cell subset-specific expression of ion channels along with their particular function in a given cell type, and the role of cancer tissue-dependent factors in the regulation of operation of these ion channels are emerging questions to be addressed in the fight against cancer disease. Answering these questions might lead to a better understanding of the immunosuppression phenomenon in cancer tissue and the development of drugs aimed at skewing the distribution of immune cell types towards killing of the tumour cells.

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.

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.

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.

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 (K(d) = 2.9 pM) and exhibits1500-fold selectivity over other ion channels assayed. It inhibits the proliferation and Ca(2+) 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.

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (T(EM)). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni²⁺ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of ¹⁵N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a K(d) of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T(EM) cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of ¹⁵N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1). Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

International audience; Animal toxins are highly reticulated and structured polypeptides that adopt a limited number of folds. In scorpion species, the most represented fold is the alpha/beta scaffold in which an helical structure is connected to an antiparallel beta-sheet by two disulfide bridges. The intimate relationship existing between peptide reticulation and folding remains poorly understood. Here, we investigated the role of disulfide bridging on the 3D structure of HsTx1, a scorpion toxin potently active on Kv1.1 and Kv1.3 channels. This toxin folds along the classical alpha/beta scaffold but belongs to a unique family of short-chain, four disulfide-bridged toxins. Removal of the fourth disulfide bridge of HsTx1 does not affect its helical structure, whereas its two-stranded beta-sheet is altered from a twisted to a nontwisted configuration. This structural change in HsTx1 is accompanied by a marked decrease in Kv1.1 and Kv1.3 current blockage, and by alterations in the toxin to channel molecular contacts. In contrast, a similar removal of the fourth disulfide bridge of Pi1, another scorpion toxin from the same structural family, has no impact on its 3D structure, pharmacology, or channel interaction. These data highlight the importance of disulfide bridging in reaching the correct bioactive conformation of some toxins.

Through a combination of fluorescence microscopy and patch-clamp analysis we have identified a striking alteration in K(+) channel expression in terminally differentiated human CCR7(-)CD45RA(-) effector memory T lymphocytes (T(EM)). Following activation, T(EM) cells expressed significantly higher levels of the voltage-gated K(+) channel Kv1.3 and lower levels of the calcium-activated K(+) channel IKCa1 than naive and central memory T cells (T(CM)). Upon repeated in vitro antigenic stimulation, naive cells differentiated into Kv1.3(high)IKCa1(low) T(EM) cells, and the potent Kv1.3-blocking sea anemone Stichodactyla helianthus peptide (ShK) suppressed proliferation of T(EM) cells without affecting naive or T(CM) lymphocytes. Thus, the Kv1.3(high)IKCa1(low) phenotype is a functional marker of activated T(EM) lymphocytes. Activated myelin-reactive T cells from patients with MS exhibited the Kv1.3(high)IKCa1(low) T(EM) phenotype, suggesting that they have undergone repeated stimulation during the course of disease; these cells may contribute to disease pathogenesis due to their ability to home to inflamed tissues and exhibit immediate effector function. The Kv1.3(high)IKCa1(low) phenotype was not seen in glutamic acid decarboxylase, insulin-peptide or ovalbumin-specific and mitogen-activated T cells from MS patients, or in myelin-specific T cells from healthy controls. Selective targeting of Kv1.3 in T(EM) cells may therefore hold therapeutic promise for MS and other T cell-mediated autoimmune diseases.

ShK, a 35-residue peptide from a sea anemone, is a potent blocker of potassium channels. Here we describe a new ShK analogue with an additional C-terminus Lys residue and amide. ShK-K-amide is a potent blocker of Kv1.3 and, in contrast to ShK and ShK-amide, is selective for Kv1.3. To understand this selectivity, we created complexes of ShK-K-amide with Kv1.3 and Kv1.1 using docking and molecular dynamics simulations, then performed umbrella sampling simulations to construct the potential of mean force of the ligand and calculate the corresponding binding free energy for the most stable configuration. The results agree well with experimental data.

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 β 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 β subunits. β1 and β3 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 β1 and β3b regulatory subunits in RA-FLS. KCa1.1 β3b subunits were expressed by 70 % of the cells and were associated with highly invasive CD44high RA-FLS, whereas minimally invasive CD44low RA-FLS and OA-FLS expressed either β1 subunit. Furthermore, we found that silencing the β3 but not the β1 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 β3b subunits as an attractive target for the therapy of RA.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease attacking principally freely-moveable joints and affecting approximately 1.3 million in the US. Recently, joint-resident fibroblast-like synoviocytes in RA (RA-FLS) have been implicated in disease pathogenesis. We have shown that KCa1.1 is the predominant potassium channel expressed by RA-FLS and by FLS from the pristane-induced arthritis (PIA) rat model of RA. Blocking KCa1.1 with paxilline or iberiotoxin or reducing its expression with siRNA inhibited the production of pro-inflammatory cytokines, chemokines, and proteases, and the invasiveness of both RA- and PIA-FLS. In contrast, the over-expression of KCa1.1 increased the invasiveness of PIA-FLS and induced the invasiveness of healthy rat FLS. These data demonstrate a crucial role of KCa1.1 in regulating the aggressive behavior of FLS during RA.We induced two models of RA in rats, moderate PIA and severe complete Freund's adjuvant collagen-induced arthritis. Treatment with the small molecule KCa1.1 blocker paxilline, starting after onset of clinical signs, significantly reduced disease severity in both models. However, paxilline can cross the blood-brain barrier and block all variants of KCa1.1 throughout the body, thereby inducing side effects that preclude its use as a therapeutic for human use without significant modification. A strategy to target KCa1.1 on RA-FLS without side effects in other tissues involves the identification of the β and γ regulatory subunits of KCa1.1 in RA-FLS through qPCR, western blotting, and patch-clamp electrophysiology. This has the potential for the development of blockers that selectively target KCa1.1 on RA-FLS and do not enter the central nervous system.

Parasites have coexisted with their human hosts for thousands of years and are known mainly for their harmful disease-causing role in humans. However, probiotic worm therapy is beneficial in human autoimmune diseases, suggesting the existence of immunomodulators in parasitic worms. By screening a cDNA library and searching genome databases we identified a family of peptides in parasitic worms that share sequence similarity and evolutionary relatedness to potassium channel-blocking peptides secreted by venomous sea anemones. AcK1, a 51-residue secreted peptide of the hookworm Ancylostoma caninum, and BmK1, the C-terminal domain of a 413-residue zinc metalloprotease from the filarial worm Brugia malayi, share structural similarity to ShK and BgK peptides from sea anemones. These peptides block cloned and native human T-cell Kv1.3 channels at nanomolar to low micromolar concentrations. BmK2, an analog of BmK1, blocks Kv1.3 with an IC50 of 2 nM and exhibits >4000-fold selectivity for Kv1.3 over Kv1.1, Kv1.2, Kv1.6, Kv3.2, KCa3.1, K2P3.1. These peptides suppress proliferation of effector memory T cells that use Kv1.3 channels to regulate membrane potential, without affecting other T cell subsets that are not dependent on Kv1.3. They inhibit cytokine production and suppress the in vivo delayed type hypersensitivity response. Our results provide a mechanistic basis for pro-biotic worm therapy in human autoimmune disease, and suggest that these or related peptides and proteins could supplant the need for worm therapy.