Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K þ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K þ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K þ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/ macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1 À/À mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.

Supplementary Figure 1 TRAM-34 treatment with 10 mg/kg initiated 2 h after reperfusion and continued for 7 days twice daily noticeably reduced infarct area following similar reductions in cerebral blood flow (control: 51.2 ± 8.2% flux reduction, n = 10; TRAM-34: 49.0 ± 7.4% flux reduction, n = 11). (A) Analysis of each affected brain slice from 2 mm to 16 mm from the frontal pole of the brain, revealed a marked reduction of TTC-defined lesion areas in all slices, which was statistically significant in the 6, 8, 10, 12 and 14-mm slices. (B) The mean infarct area was 18.8 ± 3.5% of the ipsolateral TTC + hemisphere area (n = 10) in controls and 6.6 ± 1.9% in TRAM-34 treated animals (n = 11; mean ± SEM, P = 0.007). (C) TRAM-34 treatment reduces microglia activation. Shown are means (± SEM) of ED1 + hemisphere area from the 8 and 10-mm slices from 5 animals of each group. Effects of Chronic TRAM-34 TreatmentAdult (9-11 week old) male and female rats were purchased from Charles River (Wilmington, MA) and injected i.p. for 28 days with either 20 mg/kg/d of TRAM-34 divided into two daily doses or with vehicle (n = 6 per group with 3 male and 3 female). At the end of the trial, rats were sacrificed and complete blood chemistry, hematology and necropsy performed by the Comparative Pathology Laboratory of the University of California, Davis as follows: 1. Rat were euthanized with CO 2 . 2. Whole animal weight (to mg) determined using an Ohaus analytical scale.3. Blood was collected via cardiac puncture for hematological profile (Complete blood count and differential) into EDTA coated samplette tubes. The rest of the blood will was placed in a serum separator samplette tube and separated by centrifugation at 3500 rpm. 4. The hematological profile was performed by the Comparative pathology Laboratory using a Drew Mascot 850 automated cell counter. Morphology of blood cells and WBC differential was also evaluated manually. 5. Serum Chemistry was performed on a Cobas Mira Plus chemistry analyzer. 6. Necropsies were performed and any gross lesions/changes were documented. 7. Pelage and cecal/intestinal contents was sampled and examined microscopically for parasites. 8. Liver, both kidneys, spleen, GI tract, pancreas, lung, heart, brain, bone marrow, proximal and distal lymph nodes, thymus, gonads and any lesioned organs were collected and fixed in 10% buffered formalin. Tissues were sectioned and processed to paraffin blocks. Hematoxylin and eosin stained sections from each block were examined for histologic changes by trained pathologist and any changes noted. 1) 28-Day Toxicity StudyVehicle (n = 6; 3 male, 3 female) All animals showed extra-medullary hematopoiesis in the red pulp and prominent marginal zones in the white pulp. This is a normal finding in adult rats. ‡ TRAM-34The unilateral hydronephrosis in 2 female control rats and 1 female TRAM-34 treated rat is most likely congenital in origin and has been previously seen in female rats from the same vendor. 2) 6-Months Toxicity StudyIn another trial male and female rats ...

ObjectiveInhibitors of the voltage‐gated K+ channel Kv1.3 are currently in development as immunomodulators for the treatment of autoimmune diseases. As Kv1.3 is also expressed on microglia and has been shown to be specifically up‐regulated on “M1‐like” microglia, we here tested the therapeutic hypothesis that the brain‐penetrant small‐molecule Kv1.3‐inhibitor PAP‐1 reduces secondary inflammatory damage after ischemia/reperfusion.MethodsWe studied microglial Kv1.3 expression using electrophysiology and immunohistochemistry, and evaluated PAP‐1 in hypoxia‐exposed organotypic hippocampal slices and in middle cerebral artery occlusion (MCAO) with 8 days of reperfusion in both adult male C57BL/6J mice (60 min MCAO) and adult male Wistar rats (90 min MCAO). In both models, PAP‐1 administration was started 12 h after reperfusion.ResultsWe observed Kv1.3 staining on activated microglia in ischemic infarcts in mice, rats, and humans and found higher Kv1.3 current densities in acutely isolated microglia from the infarcted hemisphere than in microglia isolated from the contralateral hemisphere of MCAO mice. PAP‐1 reduced microglia activation and increased neuronal survival in hypoxia‐exposed hippocampal slices as effectively as minocycline. In mouse MCAO, PAP‐1 dose‐dependently reduced infarct area, improved neurological deficit score, and reduced brain levels of IL‐1β and IFN‐γ without affecting IL‐10 and brain‐derived nerve growth factor (BDNF) levels or inhibiting ongoing phagocytosis. The beneficial effects on infarct area and neurological deficit score were reproduced in rats providing confirmation in a second species.InterpretationOur findings suggest that Kv1.3 constitutes a promising therapeutic target for preferentially inhibiting “M1‐like” inflammatory microglia/macrophage functions in ischemic stroke.

Cerebral edema forms in the early hours of ischemic stroke by processes involving increased transport of Na and Cl from blood into brain across an intact blood-brain barrier (BBB). Our previous studies provided evidence that the BBB Na-K-Cl cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema and infarct in rats subjected to permanent middle cerebral artery occlusion (pMCAO). More recently, we showed that BBB Na/H exchanger activity is also stimulated by hypoxia, aglycemia, and AVP. The present study was conducted to further investigate the possibility that a BBB Na/H exchanger also participates in edema formation during ischemic stroke. Sprague-Dawley rats were subjected to pMCAO and then brain edema and Na content assessed by magnetic resonance imaging diffusion-weighed imaging and magnetic resonance spectroscopy Na spectroscopy, respectively, for up to 210 minutes. We found that intravenous administration of the specific Na/H exchange inhibitor HOE-642 significantly decreased brain Na uptake and reduced cerebral edema, brain swelling, and infarct volume. These findings support the hypothesis that edema formation and brain Na uptake during the early hours of cerebral ischemia involve BBB Na/H exchanger activity as well as Na-K-Cl cotransporter activity.

Kv1.3 channels regulate the activation/proliferation of effector memory T cells and thus play a critical role in the pathogenesis of autoimmune diseases. Using a combination of immunohistochemistry, confocal microscopy, flow cytometry and electrophysiology methods we observed a significant enrichment of activated Kv1.3+ memory T cells in psoriasis plaques and synovial fluid from patients with psoriasis/psoriatic arthritis (PsA) compared to non-lesional psoriatic skin, normal skin or peripheral blood lympho-mononuclear cells. In in vitro studies performed with lesional mononuclear cells or T cells derived from skin and joints of psoriatic disease, the small molecule Kv1.3 blocker PAP-1 dose-dependently inhibited proliferation and suppressed IL-2 and IFN-γ production. To further substantiate the pathologic role of Kv1.3highTEM cells in psoriatic disease we tested whether PAP-1 is able to improve psoriatic disease pathology in the SCID mouse-psoriasis skin xenograft model. Following four weeks of daily treatment with 2% PAP-1 ointment we noticed about 50% reduction in the epidermal thickness (rete peg length) and the number of CD3+ lymphocytes/mm2 of dermis decreased by 85%. Vehicle treated and untreated plaques in contrast remained unchanged and showed no reduction in epidermis thickness and infiltrating CD3+ T cells and HLA-DR+ T cells. Based on these results we propose the development of Kv1.3 targeted topical immunotherapy for psoriasis and possibly for other inflammatory skin conditions, where effector memory T cells are involved in the pathogenesis.

Background and Purpose KCa3.1, a calcium-activated potassium channel, regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. However, the expression and physiological role of KCa3.1 in blood-brain barrier (BBB) endothelium has not been investigated. BBB endothelial cells transport Na+ and Cl− from the blood into the brain transcellularly through the cooperation of multiple co-transporters, exchangers, pumps and channels. In the early stages of cerebral ischemia, when the BBB is intact, edema formation occurs by processes involving increased BBB transcellular Na+ transport. This study evaluated whether KCa3.1 is expressed on and participates in BBB ion transport. Methods The expression of KCa3.1 on cultured cerebral microvascular endothelial cells (CMEC), isolated microvessels and brain sections was evaluated by Western blot and immunohistochemistry. Activity of KCa3.1 on CMEC was examined by K+ flux assays and patch-clamp. Magnetic resonance spectroscopy and imaging were used to measure brain Na+ uptake and edema formation in rats with focal ischemic stroke following TRAM-34 treatment. Results KCa3.1 current and channel protein were identified on bovine CMEC and freshly isolated rat microvessels. In situ KCa3.1 expression on BBB endothelium was confirmed in rat and human brain sections. TRAM-34 treatment significantly reduced Na+ uptake, and cytotoxic edema in the ischemic brain. Conclusions BBB endothelial cells exhibit KCa3.1 protein and activity and pharmacological blockade of KCa3.1 appears to provide an effective therapeutic approach for reducing cerebral edema formation in the first 3 hours of ischemic stroke.

Rationale cardiac myocyte contraction is caused by Ca2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca2+ release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca2+ release, such as Ca2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca2+-contraction coupling in live cardiac myocytes. Objective To develop a method for imaging sarcomere-level Ca2+-contraction coupling in healthy and disease-model cardiac myocytes. Methods and Results Freshly isolated cardiac myocytes were loaded with the Ca2+-indicator Fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation (SHG) from A-bands of myofibrils and two-photon fluorescence (2PF) from Fluo-4. Ca2+ signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca2+ sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca2+ activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure overload-induced cardiomyopathy. Conclusions Multi-modal SHG-2PF microscopy enables the simultaneous observation of Ca2+ release and mechanical strain at the sub-sarcomere level in living cardiac myocytes. The method benefits from the label-free nature of SHG, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca2+ indicators. SHG-2PF imaging is widely applicable to the study of Ca2+-contraction coupling and mechano-chemo-transduction in both health and disease.

Background The calcium-activated potassium channel KCa3.1 is critically involved in T cell activation, as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacological blockade would prevent the development of OAD. Methods Tracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1−/− mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120mg/kg/d). Histopathology and immunological assays were performed on postoperative days (POD) 5 or 28. Results Subepithelial T cell and macrophage infiltration on POD 5, as seen in untreated allografts, was significantly reduced in the KCa3.1−/− and TRAM-34 groups. Also, systemic Th1 activation was significantly, and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89±21% in untreated recipients to 53±26% (p=0.010) and 59±33% (p=0.032) in KCa3.1−/− and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1−/− and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1−/− animals. KCa3.1 was detected in T cells, airway epithelial cells and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes, but did not show any effect on KCa3.1−/− splenocytes. Conclusions Our findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.