Membrane expression of cloned human K_Ca3.1 in HEK-293 in inside-out patches and basic pharmacological characterization.

<p>A) From left to right: Exemplary traces of immediate activation of hK_Ca3.1-outward currents upon excision of the patch into 3 µM Ca²⁺-containing bath solution (as indicated by arrow). K_Ca-outward currents are absent in non-transfected HEK-293. Inhibition of hK_Ca3.1-outward currents by charybdotoxin (100 nM, in the pipette solution) and TRAM-34 (1 µM, in the bath solution). B) Inhibition of hK_Ca3.1 by ω3 and arachidonic acid. From left to right: Time course of inactivation of hK_Ca3.1 by docosahexaenoic acid (DHA, 10 µM), arachidonic acid (AA, 10 µM), α-linolenic acid (α-LA, µM) over time. Saturated arachidic acid (ArA, 10 µM) did not affect channel activity. C) Concentration-dependence of inhibition. Note that half of the current was inhibited by AA, DHA, and α-LA at approx. 1 µM. D) Time course of channel inactivation by two concentrations of AA, DHA, and α-LA over time. Data are means ± SEM (% inhibition of K_Ca3.1-current normalized to initial peak amplitude after patch-excision); numbers in the graphs indicate the number of inside-out experiments; *<i>P</i><0.05 vs. vehicle (Ve); One-way ANOVA and Tukey <i>post hoc</i> test.</p

5,6-EET-potentiation of K_Ca3.1 currents.

<p>A) Whole-cell current traces; from left to right: potentiation of Ca²⁺-pre-activated hK_Ca3.1 by 5,6-EET (1 µM) followed by inhibition of the current by AA (10 µM), insensitivity of the hK_Ca3.1^T250S/V275A mutant to 5,6-EET, and insensitivity of the hK_Ca3.1^T250S/V275A mutant to AA (10 µM) and TRAM-34 (1 µM). The hK_Ca3.1 currents were pre-activated by 250 nM Ca²⁺. Panel on the right: summary data. B) From left to right: Ca²⁺-pre-activation of rat endothelial rK_Ca3.1 by 3 µM Ca²⁺ and current inhibition by 14,15-EET (1 µM), larger currents in the presence of 5,6-EET (1 µM) and inhibition by AA (10 µM). Panel on right: Summary data: dependence of 5,6-EET-potentiation on the intracellular Ca²⁺. Note that at a low intracellular Ca²⁺ (0.1 µM) that is below/near the threshold for K_Ca3.1 activation, 5,6-EET did not potentiate the current. In contrast, potentiation occurred at an intracellular Ca²⁺ concentration that is near the EC₅₀ for Ca²⁺-activation of K_Ca3.1 as well as at a saturating Ca²⁺ concentration. C) DHA (1 µM) blocked Ca²⁺-pre-activated mK_Ca3.1 in murine fibroblasts. D) Pentacyclic triterpenes did not modulate murine fibroblast mK_Ca3.1 at a concentration of 1 µM. Data are means ± SEM (% inhibition of K_Ca3.1-current normalized to initial peak amplitude after establishing electrical access (by seal rupture) and stable Ca²⁺-activation of K_Ca3.1-outward currents); Numbers in the graphs indicate the number of whole-cell experiments; *<i>P</i><0.05 vs. control (peak amplitude of the K_Ca3.1-current in the respective cell); One-way ANOVA and Tukey <i>post hoc</i> test.</p

Insensitivity of hK_Ca3.1 mutants.

<p>A) Representative current traces obtained from inside-out recordings using HEK-293 expressing the hK_Ca3.1^V275A mutant. B) Summary data from experiments using the three different hK_Ca3.1 mutants and wt hK_Ca3.1. Concentration of all compounds was 10 µM. Data are means ± SEM; numbers in the graphs indicate the number of inside-out experiments. *<i>P</i><0.05 vs. wt; One-way ANOVA and Tukey <i>post hoc</i> test.</p

Chemical structures of eicosanoids, ω3, and pentacyclic triterpenes and schematic overview of blocking efficacy (decreasing from top to bottom) or non-blocking efficacy.

<p>Chemical structures of eicosanoids, ω3, and pentacyclic triterpenes and schematic overview of blocking efficacy (decreasing from top to bottom) or non-blocking efficacy.</p

Moderate antagonism of AA-mediated hK_Ca3.1-inhibtion by 5,6-EET.

<p>A) Time course of channel inhibition by 10 µM of AA in the presence of 1 µM 5,6-EET. B) Summary data of channel inhibition at 20 s after seal excision and with two concentrations (1 and 10 µM) of 5,6-EET and AA. Data are means ± SEM; numbers in the graphs indicate the number of inside-out experiments; *<i>P</i><0.05 vs. AA alone, One-way ANOVA and Tukey <i>post hoc</i> test.</p

Pharmacological modulation of KCa3.1 channels by natural phenols and NSID.

<p>Original recordings of KCa3.1 whole-cell currents in 3T3 fibroblasts are shown. Currents were activated by infusion of 1 µM Ca²⁺_free via the patch-pipette and exhibited voltage-independence and inward-rectification typical for KCa3.1. A) On left: Complete inhibition of KCa3.1 channels by caffeic acid. On right: Weak inhibition by vanillic acid. B) On left: Complete inhibition of KCa3.1 channels by flufenamic acid. On right: The structurally similar niflumic acid had no blocking activity.</p

13b and SKA-31 modulate 5-HT-induced contractions in porcine coronary artery.

<p>Data are given as mean ± SEM; n.s. not significant.</p

Lung mRNA expression levels of A) eNOS, K_Ca2.1, K_Ca2.2, K_Ca2.3, K_Ca3.1, and K_Ca1.1; B) α-Smooth muscle actin (α-SMA), collagen-1, and TGFβ.

<p>Data are given as means ±SEM, n = 7–8. Data were analyzed by two-way ANOVA and differences were considered significant when *P<0.05 from wild type, ^# P<0.05 from normoxia. Statistical interaction (£) was observed in K_Ca2.3 expression (A). C–E) Genotyping: C:Gel electrophoresis shows that polymerase chain reacton (PCR) detected the K_Ca2.3-wild type allele (wild type (+)) and the tTA allele (T) in K_Ca2.3^T/+, and K_Ca2.3^T/T. D and E: PCR detected the targeted allele in K_Ca3.1^−/+ and in K_Ca3.1^−/− as well as the wild type allele in K_Ca3.1^+/− and K_Ca3.1^+/+. A DNA ladder was used to determine products sizes.</p

Inhibition of KCa channels by 13b and TRAM-34 in freshly isolated porcine coronary artery endothelial cells.

<p>Representative whole-cell current recordings are shown. Upper panel on left: 13b-blockade of KCa currents (activated by infusion of 1 µM Ca²⁺_free via the patch-pipette; cells, n = 3). Upper panel on right: Blockade of KCa3.1 current by 1 µM TRAM-34 and blockade of the residual current (KCa2.3) by 13b (n = 4). Lower panel on left: Blockade of SKA-31-activated currents by 1 µM 13b (n = 1).</p

Inhibitory effects of natural and synthetic (poly)phenols, synthetic benzoic acids, and non-steroidal anti-inflammatory drugs on KCa3.1 channels.

<p>NT, not tested; data are given as mean ± SEM, n ≥3.</p