Small-conductance (KCa2.1-2.3) and intermediate-conductance (KCa3.1) calcium-activated K ϩ channels are critically involved in modulating calcium-signaling cascades and membrane potential in both excitable and nonexcitable cells. Activators of these channels constitute useful pharmacological tools and potential new drugs for the treatment of ataxia, epilepsy, and hypertension. Here, we used the neuroprotectant riluzole as a template for the design of KCa2/3 channel activators that are potent enough for in vivo studies.

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe -/-mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1 -/-mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe -/-mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance Ca 2+ -activated K + channel (K Ca 3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that K Ca 3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of K Ca 3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated K Ca 3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of K Ca 3.1 functions potently inhibited fibroblast proliferation by G 0 /G 1 arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of K Ca 3.1 in affected kidneys. Mice lacking K Ca 3.1 (K Ca 3.1 −/− ) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and αSMA + cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective K Ca 3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that K Ca 3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that K Ca 3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease.

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 ...

Objective— We previously demonstrated that upregulation of intermediate-conductance Ca 2+ -activated K + channels (K Ca 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K Ca 3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results— Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K Ca 3.1 blocker TRAM-34. Expression of K Ca 3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K Ca 3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K Ca 3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion— Blockade of K Ca 3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.