Carvedilol targets human K2P 3.1 (TASK1) K+ leak channels

BACKGROUND AND PURPOSE

Human K_2P3.1 (TASK1) channels represent potential targets for pharmacological management of atrial fibrillation. K_2P channels control excitability by stabilizing membrane potential and by expediting repolarization. In the heart, inhibition of K_2P currents by class III antiarrhythmic drugs results in action potential prolongation and suppression of electrical automaticity. Carvedilol exerts antiarrhythmic activity and suppresses atrial fibrillation following cardiac surgery or cardioversion. The objective of this study was to investigate acute effects of carvedilol on human K_2P3.1 (hK_2P3.1) channels.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hK_2P3.1 currents from Xenopus oocytes, Chinese hamster ovary (CHO) cells and human pulmonary artery smooth muscle cells (hPASMC).

KEY RESULTS

Carvedilol concentration-dependently inhibited hK_2P3.1 currents in Xenopus oocytes (IC₅₀= 3.8 µM) and in mammalian CHO cells (IC₅₀= 0.83 µM). In addition, carvedilol sensitivity of native I_K2P3.1 was demonstrated in hPASMC. Channels were blocked in open and closed states in frequency-dependent fashion, resulting in resting membrane potential depolarization by 7.7 mV. Carvedilol shifted the current–voltage (I–V) relationship by −6.9 mV towards hyperpolarized potentials. Open rectification, characteristic of K_2P currents, was not affected.

CONCLUSIONS AND IMPLICATIONS

The antiarrhythmic drug carvedilol targets hK_2P3.1 background channels. We propose that cardiac hK_2P3.1 current blockade may suppress electrical automaticity, prolong atrial refractoriness and contribute to the class III antiarrhythmic action in patients treated with the drug.     

Protein kinase C-independent inhibition of arterial smooth muscle K(+) channels by a diacylglycerol analogue

BACKGROUND AND PURPOSE

Analogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K⁺ and Ca²⁺ channels. We investigated whether dioctanoyl-sn-glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K⁺ currents of rat mesenteric arterial smooth muscle in a PKC-independent manner.

EXPERIMENTAL APPROACH

Conventional whole-cell and inside-out patch clamp was used to measure the inhibition of K⁺ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide.

KEY RESULTS

Mesenteric artery smooth muscle K_v currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40-fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and K_ATP currents, and again a significant proportion of these blocks occurred independently of PKC activation.

CONCLUSIONS AND IMPLICATIONS

These results show that DiC8 has a direct effect on arterial smooth muscle K⁺ channels, and this precludes its use as a PKC activator when investigating PKC-mediated effects on vascular K⁺ channels.     

TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

BACKGROUND AND PURPOSE

TASK1 (K_2P3.1) two-pore-domain K⁺ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes.

KEY RESULTS

ET-1 inhibited TASK1-mediated I_KN currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET_A (IC₅₀= 0.08 nM) and ET_B (IC₅₀= 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser³³⁶ and Ser³⁹³. Mutation of Ser³⁹³ rendered TASK1 channels insensitive to ET_A- or ET_B-mediated current inhibition. In contrast, removal of Ser³³⁶ selectively attenuated ET_A-dependent TASK1 regulation without affecting the ET_B pathway.

CONCLUSIONS AND IMPLICATIONS

ET-1 regulated vascular TASK1 currents through ET_A and ET_B receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.     

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

BACKGROUND AND PURPOSE

Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K⁺ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K_ATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K_ATP channels are also sensitive to rosiglitazone and what their sensitivities are.

EXPERIMENTAL APPROACH

K_IR6.1/SUR2B, K_IR6.2/SUR1, K_IR6.2/SUR2A, K_IR6.2/SUR2B and K_IR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

KEY RESULTS

Rosiglitazone inhibited all isoforms of K_ATP channels in excised patches and in the whole-cell configuration. Its IC₅₀ was 10 µmol·L⁻¹ for the K_IR6.1/SUR2B channel and ∼45 µmol·L⁻¹ for K_IR6.2/SURx channels. Rosiglitazone also inhibited K_IR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC₅₀= 45 µmol·L⁻¹) almost identical to that for K_IR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K_IR1.1, K_IR2.1 and K_IR4.1 channels, suggesting that the channel inhibitory effect is selective for K_IR6.x channels.

CONCLUSIONS AND IMPLICATIONS

These results suggest a novel K_ATP channel inhibitor that acts on the pore-forming K_IR6.x subunit, affecting the channel gating.

LINKED ARTICLE

This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x     

Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels

BACKGROUND AND PURPOSE

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used for the treatment of pain and inflammation. Emerging and accumulating evidence suggests that celecoxib can affect cellular targets other than COX, such as ion channels. In this study, we characterized the effects of celecoxib on K_v7 K⁺ channels and compared its effects with the well-established K_v7 channel opener retigabine.

EXPERIMENTAL APPROACH

A perforated whole-cell patch technique was used to record K_v7currents expressed in HEK 293 cells and M-type currents from rat superior cervical ganglion neurons.

KEY RESULTS

Celecoxib enhanced K_v7.2–7.4, K_v7.2/7.3 and K_v7.3/7.5 currents but inhibited K_v7.1 and K_v7.1/KCNE1 currents and these effects were concentration dependent. The IC₅₀ value for inhibition of K_v7.1 channels was approximately 4 µM and the EC₅₀ values for activation of K_v7.2–7.4, K_v7.2/K_v7.3 and K_v7.3/K_v7.5 channels were approximately 2–5 µM. The effects of celecoxib were manifested by increasing current amplitudes, shifting the voltage-dependent activation curve in a more negative direction and slowing the deactivation of K_v7 currents. 2,5-Dimethyl-celecoxib, a celecoxib analogue devoid of COX inhibition activity, has similar but greater effects on K_v7currents. K_v7.2(A235T) and K_v7.2(W236L) mutant channels, which have greatly attenuated responses to retigabine, showed a reversed response to celecoxib, from activation to inhibition.

CONCLUSIONS AND IMPLICATIONS

These results suggest that K_v7 channels are targets of celecoxib action and provide new mechanistic evidence for understanding the effects of celecoxib. They also provide a new approach to developing K_v7 modulators and for studying the structure–function relationship of K_v7 channels.     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Activation of endothelial and epithelial K(Ca) 2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

BACKGROUND AND PURPOSE

Small (K_Ca2) and intermediate (K_Ca3.1) conductance calcium-activated potassium channels (K_Ca) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of K_Ca2.3 and K_Ca3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries.

EXPERIMENTAL APPROACH

Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. K_Ca2 and K_Ca3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques.

RESULTS

While K_Ca2.3 expression was similar, K_Ca3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive K_Ca2.3 and K_Ca3.1 proteins were found in both endothelium and epithelium. K_Ca currents were present in HSAEpi cells and sensitive to the K_Ca2.3 blocker UCL1684 and the K_Ca3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the K_Ca2.3/ K_Ca3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K_Ca2 channel blocker apamin, while the K_Ca3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01–1 µM).

CONCLUSIONS AND IMPLICATIONS

K_Ca2.3 and K_Ca3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. K_Ca2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.     

Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation

BACKGROUND AND PURPOSE

Rosiglitazone is a widely used oral hypoglycaemic agent, which improves insulin resistance in type 2 diabetes. Chronic rosiglitazone treatment is associated with a number of adverse cardiac events. The present study was designed to characterize the effects of rosiglitazone on cloned K_v4.3 potassium channels.

EXPERIMENTAL APPROACH

The interaction of rosiglitazone with cloned K_v4.3 channels stably expressed in Chinese hamster ovary cells was investigated using whole-cell patch-clamp techniques.

KEY RESULTS

Rosiglitazone decreased the currents carried by K_v4.3 channels and accelerated the current inactivation, concentration-dependently, with an IC₅₀ of 24.5 µM. The association and dissociation rate constants for rosiglitazone were 1.22 µM⁻¹·s⁻¹ and 31.30 s⁻¹ respectively. Block by rosiglitazone was voltage-dependent, increasing in the voltage range for channel activation; however, no voltage dependence was found in the voltage range required for full activation. Rosiglitazone had no effect on either the deactivation kinetics or the steady-state activation of K_v4.3 channels. Rosiglitazone shifted the steady-state inactivation curves in the hyperpolarizing direction, concentration-dependently. The K_i for the interaction between rosiglitazone and the inactivated state of K_v4.3 channels was 1.49 µM, from the concentration-dependent shift in the steady-state inactivation curves. Rosiglitazone also accelerated the kinetics of the closed-state inactivation of K_v4.3 channels. Rosiglitazone did not affect either use dependence or recovery from inactivation of K_v4.3 currents.

CONCLUSION AND IMPLICATIONS

Our results indicate that rosiglitazone potently inhibits currents carried by K_v4.3 channels by interacting with these channels in the open state and by accelerating the closed-state inactivation of K_v4.3 channels.

LINKED ARTICLE

This article is commented on by Hancox, pp. 496–498 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01281.x     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation

Background and Purpose

The β-receptor antagonist carvedilol blocks a range of ion channels. K_2P2.1 (TREK1) and K_2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K_2P2.1 and K_2P10.1 channels. Second, we sought to study ATI-dependent modulation of K_2P K⁺ current sensitivity to carvedilol.

Experimental Approach

Using standard electrophysiological techniques, we recorded currents from wild-type and mutant K_2P2.1 and K_2P10.1 channels in Xenopus oocytes and HEK 293 cells.

Key Results

Carvedilol concentration-dependently inhibited K_2P2.1 channels (IC_50,oocytes = 20.3 μM; IC_50,HEK = 1.6 μM) and this inhibition was frequency-independent. When K_2P2.1 isoforms generated by ATI were studied separately in oocytes, the IC₅₀ value for carvedilol inhibition of full-length channels (16.5 μM) was almost 5-fold less than that for the truncated channel variant (IC₅₀ = 79.0 μM). Similarly, the related K_2P10.1 channels were blocked by carvedilol (IC_50,oocytes = 24.0 μM; IC_50,HEK = 7.6 μM) and subject to ATI-dependent modulation of drug sensitivity.

Conclusions and Implications

Carvedilol targets K_2P2.1 and K_2P10.1 K⁺ channels. This previously unrecognized mechanism supports a general role of cardiac K_2P channels as antiarrhythmic drug targets. Furthermore, the work reveals that the sensitivity of the cardiac ion channels K_2P2.1 and K_2P10.1 to block was modulated by alternative mRNA translation initiation.     

Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels

Background and purpose:

Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John''s Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels.

Experimental approach:

We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na⁺, I_A and I_K currents.

Key results:

Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I_A and delayed rectifier I_K potassium currents. In contrast, hypericin exerted no significant effect on both Na⁺ peak current and its decay kinetics.

Conclusions and implications:

Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I_A and I_K currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.     

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Background and purpose:

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl⁻ channels (CaCCs) and large conductance, calcium-gated K⁺ channels (K_Ca1.1). The goal of the present study was to ascertain whether blockers of K_Ca1.1 inhibited calcium-activated Cl⁻ currents (I_ClCa) and if the pharmacological overlap between K_Ca1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K⁺ channels.

Experimental approaches:

Whole-cell Cl⁻ and K⁺ currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca²⁺].

Key results:

The selective K_Ca1.1 blocker paxilline (1 µM) inhibited I_ClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I_ClCa by ∼20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca²⁺-binding domain of murine K_Ca1.1 had no effect on I_ClCa while inhibiting spontaneous K_Ca1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K⁺ channels (K_Ca2.1 and K_Ca2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on I_ClCa.

Conclusions and implications:

These data show that the selective K_Ca1.1 blockers also reduce I_ClCa considerably. However, the pharmacological overlap that exists between CaCCs and K_Ca1.1 does not extend to the calcium-binding domain or to other calcium-gated K⁺ channels.     

The SK3/K(Ca)2.3 potassium channel is a new cellular target for edelfosine

BACKGROUND AND PURPOSE

The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function.

EXPERIMENTAL APPROACH

Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and ¹²⁵I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca²⁺-activated K⁺ channel, SK3/K_Ca2.3.

KEY RESULTS

Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/K_Ca2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit ¹²⁵I-Apamin binding to this SK_Ca channel; rather, it reduced the calcium sensitivity of SK3/K_Ca2.3 channel and dramatically decreased intracellular Ca²⁺ concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SK_Ca channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/K_Ca2.3 protein in a SK3/K_Ca2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine.

CONCLUSIONS AND IMPLICATIONS

Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/K_Ca2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.     

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

BACKGROUND AND PURPOSE

The compound NS5806 increases the transient outward current (I_to) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I_to is thought to be mediated by K_V4.3 and various ancillary proteins, yet, the exact subunit composition of I_to channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I_to channel subunits and other potassium channels.

EXPERIMENTAL APPROACH

Cloned K_V4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K_V1.4 in Xenopus laevis oocytes or CHO-K1 cells.

KEY RESULTS

NS5806 increased K_V4.3/KChIP2 peak current amplitudes with an EC₅₀ of 5.3 ± 1.5µM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K_V4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K_V4.2 was similar to that on K_V4.3, and current decay was only slowed in presence of KChIP2. However, for K_V4.1, the slowing of current decay by NS5806 was independent of KChIP2. K_V1.4 was strongly inhibited by 10 µM NS5806 and K_V1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K_V4.3/KChIP2/DPP6 with K_V1.4 in oocytes could reproduce those on cardiac I_to in canine ventricular myocytes. K_V7.1, K_V11.1 and K_ir2 currents were unaffected by NS5806.

CONCLUSION AND IMPLICATIONS

NS5806 modulated K_V4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I_to.     

Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

BACKGROUND AND PURPOSE

Controlling vascular tone involves K⁺ efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K_Ca2.3 and K_Ca3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.

EXPERIMENTAL APPROACH

Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K_Ca2.3 and K_Ca3.1 expression by immunohistochemistry and RT-PCR. K_Ca currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K_Ca3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.

KEY RESULTS

K_Ca3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K_Ca2.3 and K_Ca3.1 mRNA expression. With 200 nM [Ca²⁺]_i, the K_Ca2.1-2.3/K_Ca3.1 opener NS309 increased whole-cell currents. CyPPA, a K_Ca2.2/K_Ca2.3 opener, was without effect. With 1 µM [Ca²⁺]_i, the K_Ca3.1 inhibitor TRAM-34 reduced currents whereas apamin (K_Ca2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K_Ca3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K_Ca3.1 and the large-conductance K_Ca channel, K_Ca1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.

CONCLUSION AND IMPLICATIONS

Astrocytes express functional K_Ca3.1 channels, and these contribute to neurovascular coupling.

LINKED ARTICLES

This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3     

Levosimendan induces NO production through p38 MAPK,ERK and Akt in porcine coronary endothelial cells: role for mitochondrial KATP channel

Background and purpose:

Levosimendan acts as a vasodilator through the opening of ATP-sensitive K⁺ channels (K_ATP) channels. Moreover, the coronary vasodilatation caused by levosimendan in anaesthetized pigs has recently been found to be abolished by the nitric oxide synthase (NOS) inhibitor N^ω-nitro-L-arginine methyl ester, indicating that nitric oxide (NO) has a role in the vascular effects of levosimendan. However, the intracellular pathway leading to NO production caused by levosimendan has not yet been investigated. Thus, the purpose of the present study was to examine the effects of levosimendan on NO production and to evaluate the intracellular signalling pathway involved.

Experimental approach:

In porcine coronary endothelial cells (CEC), the release of NO in response to levosimendan was examined in the presence and absence of N^ω-nitro-L-arginine methyl ester, an adenylyl cyclase inhibitor, K_ATP channel agonists and antagonists, and inhibitors of intracellular protein kinases. In addition, the role of Akt, ERK, p38 and eNOS was investigated through Western blot analysis.

Key results:

Levosimendan caused a concentration-dependent and K⁺-related increase of NO production. This effect was amplified by the mitochondrial K_ATP channel agonist, but not by the selective plasma membrane K_ATP channel agonist. The response of CEC to levosimendan was prevented by the K_ATP channel blockers, the adenylyl cyclase inhibitor and the Akt, ERK, p38 inhibitors. Western blot analysis showed that phosphorylation of the above kinases lead to eNOS activation.

Conclusions and implications:

In CEC levosimendan induced eNOS-dependent NO production through Akt, ERK and p38. This intracellular pathway is associated with the opening of mitochondrial K_ATP channels and involves cAMP.     

Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes

Aim:

To study the effects of Na⁺ channel blocker flecainide and L-type Ca²⁺ channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K⁺ channel.

Methods:

fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K⁺ currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion.

Results:

fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of −41.74 mV and a slow recovery from inactivation (τ=1.90 s at −90 mV). Flecainide and verapamil blocked the currents with IC₅₀ values of 512.29±56.92 and 260.71±18.50 μmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve.

Conclusion:

The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K⁺ channel blockers to treat arrhythmia.     

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

BACKGROUND AND PURPOSE

Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

EXPERIMENTAL APPROACH

Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K_IR3 and K_V7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na⁺, Ca²⁺, inward rectifier K⁺, K_V7 K⁺, and TRPV1 channels, as well as GABA_A, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

KEY RESULTS

Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na⁺ or Ca²⁺ channels, inward rectifier K⁺ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K_V7 K⁺ channels to more negative potentials and the gating of GABA_A receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K_V7 channels and GABA_A receptors was comparable, whereas in hippocampal neurons the effects on K_V7 channels were more pronounced.

CONCLUSIONS AND IMPLICATIONS

These results indicate that flupirtine exerts its analgesic action by acting on both GABA_A receptors and K_V7 channels.     

Ca2+/calcineurin regulation of cloned vascular KATP channels: crosstalk with the protein kinase A pathway

Background and purpose:

Vascular ATP-sensitive potassium (K_ATP) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca²⁺-dependent phosphatase, inhibits K_ATP channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA.

Experimental approach:

Whole-cell recordings were made in HEK-293 cells stably expressing the vascular K_ATP channel (K_IR6.1/SUR2B). The effect of intracellular Ca²⁺ and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined.

Key results:

Constitutively active calcineurin Aα but not Aβ significantly attenuated K_ATP currents activated by low intracellular Ca²⁺, whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal K_ATP currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca²⁺ (equivalent to increasing calcineurin activity), almost completely inhibited K_ATP channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA.

Conclusions and implications:

Calcineurin Aα regulates K_IR6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the K_ATP channel.British Journal of Pharmacology (2009) 157, 554–564; doi:10.1111/j.1476-5381.2009.00221.x; published online 7 May 2009This article is commented on by Tammaro, pp. 551–553 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009