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The voltage-gated K+channels Kv7.2 and Kv7.3 are located at the axon initial segment (AIS) and exert strong control over action potential generation. Therefore, changes in their localization or cell surface numbers are likely to influence neuronal signaling. However, nothing is known about the cell surface dynamics of Kv7.2/7.3 at steady state or during short-term neuronal stimulation. This is primarily attributable to their membrane topology, which hampers extracellular epitope tagging. Here we circumvent this limitation by fusing an extra phluorin-tagged helix to the N terminus of human Kv7.3. This seven transmembrane chimera, named super ecliptic phluorin (SEP)–TAC-7.3, functions and traffics as a wild-type (WT) channel. We expressed SEP–TAC-7.3 in dissociated rat hippocampal neurons to examine the lateral mobility, surface numbers, and localization of AIS Kv7.2/7.3 heteromers using live imaging. We discovered that they are extraordinarily stable and exhibit a very low surface mobility both during steady state and neuronal stimulation. In the latter case, we also found that neither localization nor cell surface numbers were changed. However, at high glutamate loads, we observed a rapid irreversible endocytosis of Kv7.2/7.3, which required the activation of NR2B-containing NMDA receptors, Ca2+influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABAAreceptors, were also endocytosed. In conclusion, we have, for the first time, characterized the cell surface dynamics of a full-length Kv7 channel using a novel chimeric strategy. This approach is likely also applicable to other Kv channels and thus of value for the additional characterization of this ion channel subfamily.SIGNIFICANCE STATEMENTThe voltage-gated K+channels Kv7.2 and Kv7.3 exert strong control over action potential generation, but little is known about their cell surface dynamics. Using a novel phluorin-based approach, we here show that these channels are highly stable at steady state and different types of neuronal stimulation. However, at high glutamate loads, they undergo a rapid calpain-dependent endocytosis that likely represents an early response during excitotoxic states.

Aims The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle. Methods and results Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N 6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation. Conclusion Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work. © 2011 Elsevier Ltd.

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1–50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC50 of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2–5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.

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(V)4.3 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(V)4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K(V)1.4 in Xenopus laevis oocytes or CHO-K1 cells. Key results NS5806 increased K(V)4.3/KChIP2 peak current amplitudes with an EC(50) of 5.3 +/- 1.5microM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K(V)4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K(V)4.2 was similar to that on K(V)4.3, and current decay was only slowed in presence of KChIP2. However, for K(V)4.1, the slowing of current decay by NS5806 was independent of KChIP2. K(V)1.4 was strongly inhibited by 10 microM NS5806 and K(V)1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K(V)4.3/KChIP2/DPP6 with K(V)1.4 in oocytes could reproduce those on cardiac I(to) in canine ventricular myocytes. K(V)7.1, K(V)11.1 and K(ir)2 currents were unaffected by NS5806. Conclusion and implications NS5806 modulated K(V)4 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).

The cardiac action potential is the result of an orchestrated function of a number of different ion channels. Action potential repolarisation in humans relies on three potassium current components named I(Kr), I(Ks) and I(K1) with party overlapping functions. The ion channel alpha-subunits conducting these currents are hERG1 (Kv11.1), KCNQ1 (Kv7.1) and Kir2.1. Loss-of-function in any of these currents can result in long QT syndrome. Long QT is a pro-arrhythmic disease with increased risk of developing lethal ventricular arrhythmias such as Torsade de Pointes and ventricular fibrillation. In addition to congenital long QT, acquired long QT can also constitute a safety risk. Especially unintended inhibition of the hERG1 channel constitutes a major concern in the development of new drugs. Based on this knowledge is has been speculated whether activation of the hERG1 channel could be anti-arrhythmic and thereby constitute a new principle in treatment of cardiac arrhythmogenic disorders. The first hERG1 channel agonist was reported in 2005 and a limited number of such compounds are now available. In the present text we review results obtained by hERG1 channel activation in a number of cardiac relevant settings from in vitro to in vivo. It is demonstrated how the principle of hERG1 channel activation under certain circumstances can constitute a new anti-arrhythmogenic principle. Finally, important conceptual differences between the short QT syndrome and the hERG1 channel activation, are evaluated.

Kir3.4 and Kir3.1 potassium channel subunits mediate the acetylcholine induced inwardly rectifying current I(KACh) in the heart. We found a glycine to arginine substitution in codon 247 of Kir3.4 in a patient with a single episode of atrial fibrillation (AF). Expression in Xenopus laevis oocytes and two-electrode voltage-clamp revealed that Kir3.4-G247R basal current was reduced compared to wild-type Kir3.4 and co-expression with the muscarinic acetylcholine receptor type 2 showed that also the acetylcholine induced current was severely reduced in Kir3.4-G247R, indicating that the mutation interfered with activation by the stimulatory G betagamma-subunits. Co-expression of Kir3.4-G247R with wild-type Kir3.4 or Kir3.1 had a compensating effect on both basal current levels and the response to muscarinic stimulation suggesting the function of Kir3.4-G247R is compensated in vivo. This may explain the lack of clear clinical manifestations and further studies are necessary to elucidate if mutations in Kir3.4 are predisposing AF.

Large-conductance Ca(2+)- and voltage-activated K(+) channels (Kca1.1/BK/MaxiK) are widely expressed ion channels. They provide a Ca(2+)-dependent feedback mechanism for the regulation of various body functions such as blood flow, neurotransmitter release, uresis, and immunity. In addition, a mitochondrial K(+) channel with KCa1.1-resembling properties has been found in the heart, where it may be involved in regulation of energy consumption. In the present study, the effect of a novel NeuroSearch compound, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea (NS11021), was investigated on cloned KCa1.1 expressed in Xenopus laevis oocytes and mammalian cells using electrophysiological methods. NS11021 at concentrations above 0.3 microM activated KCa1.1 in a concentration-dependent manner by parallel-shifting the channel activation curves to more negative potentials. Single-channel analysis revealed that NS11021 increased the open probability of the channel by altering gating kinetics without affecting the single-channel conductance. NS11021 (10 microM) influenced neither a number of cloned Kv channels nor endogenous Na(+) and Ca(2+) channels (L- and T-type) in guinea pig cardiac myocytes. In conclusion, NS11021 is a novel KCa1.1 channel activator with better specificity and a 10 times higher potency compared with the most broadly applied KCa1.1 opener, NS1619. Thus, NS11021 might be a valuable tool compound when addressing the physiological and pathophysiological roles of KCa1.1 channels.

In neuronal tissue, KCNQ2-5 channels conduct the physiologically important M-current. In some neurones, the M-current may in addition be conducted partly by ERG potassium channels, which have widely overlapping expression with the KCNQ channel subunits. XE991 and linopiridine are known to be standard KCNQ potassium channel blockers. These compounds have been used in many different tissues as specific pharmacological tools to discern native currents conducted by KCNQ channels from other potassium currents. In this article, we demonstrate that ERG1-2 channels are also reversibly inhibited by XE991 in the micromolar range (EC(50) 107 microM for ERG1). The effect has been characterized in Xenopus laevis oocytes expressing ERG1-2 and in the mammalian HEK293 cell line stably expressing ERG1 channels. The IC(50) values for block of KCNQ channels by XE991 range 1-65 microM. In conclusion, great care should be taken when choosing the concentration of XE991 to use for experiments on native potassium channels or animal studies in order to be able to conclude on selective KCNQ channel-mediated effects.

The mammalian Kv4.3 potassium channel is a fast activating and inactivating K+ channel widely distributed in mammalian tissues. Kv4.3 is the major component of various physiologically important currents ranging from A-type currents in the CNS to the transient outward potassium conductance in the heart (I(to)). Here we show that the KCNE3 beta-subunit has a strong inhibitory effect on current conducted by heterologously expressed Kv4.3 channels. KCNE3 reduces the Kv4.3 current amplitude, and it slows down the channel activation and inactivation as well as the recovery from inactivation. KCNE3 also inhibits currents generated by Kv4.3 in complex with the accessory subunit KChIP2. We find the inhibitory effect of KCNE3 to be specific for Kv4.3 within the Kv4 channel family. Kv4.3 has previously been shown to interact with a number of beta-subunits, but none of the described subunit-interactions exert an inhibitory effect on the Kv4.3 current.

Within the field of new antiarrhythmic compounds, the interesting idea of activating human ether-a-go-go-related gene (HERG1) potassium channels has recently been introduced. Potentially, drugs that increase HERG1 channel activity will augment the repolarizing current of the cardiac myocytes and stabilize the diastolic interval. This may make the myocardium more resistant to events that cause arrhythmias. We here present the compound N-(4-bromo-2-(1H-tetrazol-5-yl)-phenyl)-N'-(3'-trifluoromethylphenyl)urea (NS3623), which has the ability to activate HERG1 channels expressed in Xenopus laevis oocytes with an EC50 value of 79.4 microM. Exposure of HERG1 channels to NS3623 affects the voltage-dependent release from inactivation, resulting in a half-inactivation voltage that is rightward-shifted by 17.7 mV. Moreover, the compound affects the time constant of inactivation, leading to a slower onset of inactivation of the macroscopic HERG1 currents. We also characterized the ability of NS3623 to increase the activity of different mutated HERG1 channels. The mutants S620T and S631A are severely compromised in their ability to inactivate. Application of NS3623 to any of these two mutants did not result in increased HERG1 current. In contrast, application of NS3623 to the mutant F656M increased HERG1 current to a larger extent than what was observed with wild-type HERG1 channels. Because the amino acid F656 is essential for high-affinity inhibition of HERG1 channels, it is concluded that NS3623 has a dual mode of action, being both an activator and an inhibitor of HERG1 channels. Finally, we show that NS3623 has the ability to shorten action potential durations in guinea pig papillary muscle.

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump γ-subunit. Originally it was suggested that CHIF could induce the expression of the IKs current when expressed in Xenopus laevis oocytes, but recently CHIF has attracted attention as a modulatory subunit of the Na,K-pump. In renal and intestinal epithelia, the expression of CHIF is dramatically up-regulated in response to aldosterone stimulation, and regulation of epithelial ion channels by CHIF is an attractive hypothesis. To study a potential regulatory effect of the CHIF subunit on KCNQ1 channels, co-expression experiments were performed in Xenopus laevis oocytes and mammalian CHO-K1 cells. Electrophysiological characterization was obtained by two-electrode voltage-clamp and patch-clamp, respectively. In both expression systems, we find that CHIF drastically modulates the KCNQ1 current; in the presence of CHIF, the KCNQ1 channels open at all membrane potentials. Thereby, CHIF is the first accessory subunit shown to be capable of modulating both the Na,K-pump and an ion channel. To find a possible physiological function of the constitutively open KCNQ1/CHIF complex, the precise localization of KCNQ1 and CHIF in distal colon and kidney from control and salt-depleted rats was determined by confocal microscopy. However, in these tissues, we did not detect an obvious overlap in expression between KCNQ1 and CHIF. In conclusion, the hormone-regulated subunit CHIF modulates the voltage sensitivity of the KCNQ channels, but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking.

The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased repolarization current could be a new antiarrhythmic principle, because it possibly would attenuate afterdepolarizations, ischemic leak currents, and reentry phenomena. Repolarization of the cardiac myocytes is crucially dependent on the late rapid delayed rectifier current (I(Kr)) conducted by ether-a-go-go-related gene (ERG) potassium channels. We have developed the diphenylurea compound 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and tested whether this small organic molecule could increase the activity of human ERG (HERG) channels expressed heterologously. In Xenopus laevis oocytes, NS1643 increased both steady-state and tail current at all voltages tested. The EC(50) value for HERG channel activation was 10.5 microM. These results were reproduced on HERG channels expressed in mammalian human embryonic kidney 293 cells. In guinea pig cardiomyocytes, studied by patch clamp, application of 10 microM NS1643 activated I(Kr) and significantly decreased the action potential duration to 65% of the control values. The effect could be reverted by application of the specific HERG channel inhibitor 4'-[[1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidinyl]carbonyl]-methanesulfonanilide (E-4031) at 100 nM. Application of NS1643 also resulted in a prolonged postrepolarization refractory time. Finally, cardiomyocytes exposed to NS1643 resisted reactivation by small depolarizing currents mimicking early afterdepolarizations. In conclusion, HERG channel activation by small molecules such as NS1643 increases the repolarization reserve and presents an interesting new antiarrhythmic approach.

We have identified and characterized the compound NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) as a potent activator of human Ca2+-activated K+ channels of SK and IK types, whereas it is devoid of effect on BK type channels. IK- and SK-channels have previously been reported to be activated by the benzimidazolinone, 1-EBIO and more potently by its dichloronated-analogue, DC-EBIO. NS309 is at least 1000 times more potent than 1-EBIO and at least 30 times more potent than DC-EBIO when the compounds are compared on the same cell.

KCNQ4 channels expressed in HEK 293 cells are sensitive to cell volume changes, being activated by swelling and inhibited by shrinkage, respectively. The KCNQ4 channels contribute significantly to the regulatory volume decrease (RVD) process following cell swelling. Under isoosmotic conditions, the KCNQ4 channel activity is modulated by protein kinases A and C, G protein activation, and a reduction in the intracellular Ca2+ concentration, but these signalling pathways are not responsible for the increased channel activity during cell swelling.

The function of the KCNE5 (KCNE1-like) protein has not previously been described. Here we show that KCNE5 induces both a time- and voltage-dependent modulation of the KCNQ1 current. Interaction of the KCNQ1 channel with KCNE5 shifted the voltage activation curve of KCNQ1 by more than 140 mV in the positive direction. The activation threshold of the KCNQ1+KCNE5 complex was +40 mV and the midpoint of activation was +116 mV. The KCNQ1+KCNE5 current activated slowly and deactivated rapidly as compared to the KCNQ1+KCNE1 at 22 degrees C; however, at physiological temperature, the activation time constant of the KCNQ1+KCNE5 current decreased fivefold, thus exceeding the activation rate of the KCNQ1+KCNE1 current. The KCNE5 subunit is specific for the KCNQ1 channel, as none of other members of the KCNQ-family or the human ether a-go-go related channel (hERG1) was affected by KCNE5. Four residues in the transmembrane domain of the KCNE5 protein were found to be important for the control of the voltage-dependent activation of the KCNQ1 current. We speculate that since KCNE5 is expressed in cardiac tissue it may here along with the KCNE1 beta-subunit regulate KCNQ1 channels. It is possible that KCNE5 shapes the I(Ks) current in certain parts of the mammalian heart.

The effect of changes in pH on the gating properties of the cloned human intermediate-conductance, Ca2+-activated K+ channel (hIK) was studied using the patch-clamp technique. Multi-channel inside-out recordings of patches from HEK-293 cells stably expressing hIK channels revealed that the channel activity is modulated by changes in intracellular pH (pHi). Changes in extracellular pH (pHo) in the range from pH 6.0 to 8.2 did not affect the hIK whole-cell current. Intracellular acidification gradually decreased the activity of the hIK channel, approaching zero activity at pHi 6.0. Decreasing pHi altered neither the conductance nor the inward rectification of hIK channels. The proton-induced inhibition of the multi-channel hIK patch current could not be counteracted by increasing the cytosolic Ca2+ concentration to 30 microM. The molecular sensory mechanism underlying the proton-induced modulation of hIK gating is at present unknown.

Three genes encode the small-conductance Ca(2+)-activated K(+) channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole-cell patch clamp recordings. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC(50) values of 3.3 nM and 83 pM, respectively. The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC(50) value of 80 nM and rSK2 at 287 pM. The potent small molecule blockers showed little differentiation between the channel subtypes. The bis-quinolinium cyclophane UCL 1684 blocked hSK1 with an IC(50) value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC(50) values of 444 nM and 162 nM, respectively. The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. The GABA(A) receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC(50) values of 15 and 25 microM when measured at +80 mV. In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin-insensitive afterhyperpolarization of neurones is mediated by hSK1.

Modulation of the cloned human intermediate-conductance Ca(2+)-activated K(+) channel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stably expressing the hIK channels. In whole-cell studies, intracellular concentrations of free Ca(2+) were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to approximately 300 pA/pF without reaching saturation even at the highest Ca(2+) concentration tested (300 nM). In the presence of EBIO (100 microM), the Ca(2+)-activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca(2+). In current-clamp, steeply Ca(2+)-dependent membrane potentials were recorded and the cells gradually hyperpolarised from -20 to -85 mV when Ca(2+) was augmented from 0 to 300 nM. EBIO strongly hyperpolarised cells buffered at intermediate Ca(2+) concentrations. In contrast, no effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca(2+) (V(m) close to E(K)). Without Ca(2+), EBIO-induced hyperpolarisations were not obtainable, indicating an obligatory Ca(2+)-dependent mechanism of action. When applied to inside-out patches, EBIO exerted a Ca(2+)-dependent increase in the single-channel open-state probability, showing that the compound modulates hIK channels by a direct action on the alpha-subunit or on a closely associated protein. In conclusion, EBIO activates hIK channels in whole-cell and inside-out patches by a direct mechanism, which requires the presence of internal Ca(2+).

This report presents the results of a patch-clamp study examining the role of intracellular ATP in the regulation of endothelial inward rectifier K+ channels. Administration of ATP to the cytosolic surface of inside-out patches reversibly activated the K+ channel within seconds. ATP (1 mM) increased the mean open probability by a factor of 3.5, primarily by increasing the number of openings. Administration of the nonhydrolyzable ATP analogue ATP-gamma-S failed to modulate channel activity. Inhibition of ATP synthesis by administration of cyanide plus iodoacetate resulted in channel closure within 1 to 6 minutes. In experiments in which ATP was coadministered with the metabolic blockers, the channel activity continued unchanged for up to 30 minutes, but when ATP was removed, the activity rapidly decayed. We propose that normal functioning of the inward rectifier K+ channel is ATP dependent. Phosphorylation of the channel molecule is probably essential for maintaining activity.

We sought to explore the distribution pattern of Na+ channels across ventricular wall, and to determine its functional correlates, in the guinea pig heart. Voltage-dependent Na+ channel (Nav) protein expression levels were measured in transmural samples of ventricular tissue by Western blotting. Isolated, perfused heart preparations were used to record monophasic action potentials and volume-conducted ECG, and to measure effective refractory periods (ERPs) and pacing thresholds, in order to assess excitability, electrical restitution kinetics, and susceptibility to stimulation-evoked tachyarrhythmias at epicardial and endocardial stimulation sites. In both ventricular chambers, Nav protein expression was higher at endocardium than epicardium, with midmyocardial layers showing intermediate expression levels. Endocardial stimulation sites showed higher excitability, as evidenced by lower pacing thresholds during regular stimulation and downward displacement of the strength-interval curve reconstructed after extrasystolic stimulation compared with epicardium. ERP restitution assessed over a wide range of pacing rates showed greater maximal slope and faster kinetics at endocardial than epicardial stimulation sites. Flecainide, a Na+ channel blocker, reduced the maximal ERP restitution slope, slowed restitution kinetics, and eliminated epicardial-to-endocardial difference in dynamics of electrical restitution. Greater excitability and steeper electrical restitution have been associated with greater arrhythmic susceptibility of endocardium than epicardium, as assessed by measuring ventricular fibrillation threshold, inducibility of tachyarrhythmias by rapid cardiac pacing, and the magnitude of stimulation-evoked repolarization alternans. In conclusion, higher Na+ channel expression levels may contribute to greater excitability, steeper electrical restitution slopes and faster restitution kinetics, and greater susceptibility to stimulation-evoked tachyarrhythmias at endocardium than epicardium in the guinea pig heart.

Background The repolarizing cardiac rapid delayed rectifier current, I(Kr), is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I(Kr). Marked heterogeneity in the kinetic properties of native I(Kr) has been described. We hypothesized that the heterogeneity of native I(Kr) can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD) and restitution. Methodology/principal findings The results show that the heterogeneity of native I(Kr) can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased. Conclusions/significance Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I(Kr) kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I(Kr). Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.

KCNQ1 and its β-subunit KCNE1 form the delayed rectifier potassium current IKs, playing an important role in repolarisation of the cardiac tissue and in water and salt transport across epithelial tissues. In the heart IKs is partly responsible for terminating the cardiac action potential. Malfunctions in this channel can result in arrhythmias leading to cardiac arrest.In heart physiology proper function and regulation of IKs current is essential. It has been reported that one of the mechanisms controlling the membrane density of KCNQ1 channels is mediated by ubiquitylation. IKs was shown to be down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases and this interaction was dependent on the PY-motif on the C-terminal of KCNQ1. Recently it was also discovered that epithelial sodium channel ENaC is regulated by the reverse process - de-ubiquitylation, mediated by an enzyme USP2 (ubiquitin-specific protease 2), which is one of the best described de-ubiquitylases. Therefore the aim of the work was to investigate whether a similar mechanism is valid for KCNQ1/E1 channel complex.The effect of USP2-mediated de-ubiquitylation on IKs channel was investigated using electrophysiology and biochemistry. We observed that when KCNQ1/E1 was co-expressed with USP2-45 or USP2-69 isoform and Nedd4-2 in oocytes, USP2 counteracted the Nedd4-2-specific down-regulation of IKs. It resulted in a rescue of the current amplitude, which was then comparable to the one of IKs expressed alone. Biochemical studies of transfected HEK293 cells confirmed this observation as both total and surface expressed KCNQ1 protein was more abundant when co-expressed with USP2-45/-69 and Nedd4-2 as compared to Nedd4-2 alone. Co-immunoprecipitation assay suggested that USP2 can bind to KCNQ1 independently of the PY-motif and the presence of Nedd4-2.These results point towards an interplay between ubiquitylating enzymes and de-ubiquitylases acting on IKs channel complex in vitro.

Objective: NS5806 activates the transient outward potassium current (Ito) in canine ventricular cells. We compared the effects of NS5806 on canine ventricular versus atrial tissues and myocytes. Methods: NS5806 (10 μM) was evaluated in arterially-perfused canine right atrial and left ventricular wedges. Atrial and ventricular epi- and endocardial cells were isolated by enzymatic dissociation. Current and voltage-clamp recordings were made in the absence and presence of NS5806. Results: In ventricular wedges NS5806 increased phase 1 repolarization in epi- and midmyocardial cells. A minor effect on conduction and upstroke velocity also was observed. In contrast, application of NS5806 to atrial preparations slowed upstroke velocity and reduced excitability, consistent with sodium channel block. In ventricular myocytes, NS5806 increased the magnitude of Ito by 80% and 16% in epi and endo, respectively (at +40 mV). In atrial myocytes, NS5806 increased peak Ito by 25% and had no effect on the sustained pedestal current, IKur. INa density in atrial myocytes was nearly 100% greater than in endocardial myocytes. NS5806 caused a negative shift in steady-state mid-inactivation (V1/2) for both cell types (73.9±0.27 to −77.3±0.21mV for endocardial and −82.6±0.12 to −85.1±0.11mV for atrial cells). The shift in V1/2 resulted in a reduction of INa in both cell types. However, the more negative V1/2 in atrial cells suggests that atrial cells lose excitability at more depolarized voltages than endocardial cells which may explain the greater reduction of excitability in atrial vs ventricular wedges by NS5806. Conclusion: NS5806 produces a prominent augmentation of Ito with little effect on INa in the ventricles, but a potent inhibition of INa with little augmentation of Ito in atria.

Single-channel currents of an anionic channel in the plasma membrane of cultured bovine aortic endothelial cells have been recorded with the patch-clamp technique. The channel is selective for chloride over cations, and has an average single channel conductance of 382 picosiemens in symmetric 140 millimoles of chloride. In addition to the main conductance state it shows well-defined subconductance states of about 50, 100, 150 and 200 picosiemens. The channel is very active at membrane potentials close to 0 mV, but steps to either positive or negative membrane potentials above +/- 20 millivolt lead to a rapid inactivation of the channel. Changes in the concentrations of free calcium or adenosine tri-phosphate on the cytosolic surface do not influence channel activity. The chloride channel rarely opens at resting membrane potential, but it may help repolarize endothelial cells following depolarizing stimuli.

Background: The Brugada syndrome (BrS) is characterized by elevated ST segments in the right precordial leads, ventricular tachycardia and sudden death. The syndrome has been linked to loss-of-function of sodium and calcium channels, however the transient outward potassium current (Ito) is thought to be central in the pathogenesis of BrS. We assessed the effects of Ito augmentation in a mammalian model using a novel activator of Ito, NS5806. Methods and Results: Voltage-clamp experiments were performed on midmyocardial cells isolated from the canine left ventricular wall. At 40mV NS5806 (10 μM) increased peak Ito by 79±4 % and the time-course of inactivation was slowed (from Tau=12.6±3.2 ms to 20.3±2.9 ms). We next assessed the effect of increased Ito in the development of BrS phenotype using canine ventricular wedge preparations. NS5806 increased the epicardial action potential (AP) phase 1 magnitude, whereas the APs of endocardial cells were largely unaffected. The accentuated epicardial notch was associated with an accentuated J-wave on the ECG. Loss of the epicardial AP dome at some sites but not others, led to development of phase-2-reentry and polymorphic ventricular tachycardia. NS5806 was able to induce the BrS phenotype in wedges from both right and left ventricles of the canine heart; at 15 μM NS5806 BrS developed in 4/6 right ventricular preparations compared to 2/10 left ventricular preparations. Conclusion: The Ito agonist NS5806 recapitulates the electrographic and arrhythmic manifestation of BrS, providing evidence in support of its pivotal role in the genesis of the disease. Our findings suggest that a genetic defect leading to a prominent gain of function of Ito could explain variants of BrS in which ST segment elevation are evident in both right and left ECG leads.

Background: In human and canine ventricles the transient outward current (Ito) is mediated by Kv4.3 and various accessory proteins. However, the exact molecular composition constituting Ito is still debated. We used the NeuroSearch compound NS5806 known to increase Ito in canine ventricular myocytes as a tool to investigate the molecular components comprising Ito. Methods and Results: The effect of NS5806 was investigated on cloned Kv4.3 channels heterologously expressed together with various β-subunits in Xenopus laevis oocytes. NS5806 was found to inhibit Kv4.3 currents with an IC50 of 6.2 μM, whereas it caused a dose-dependent increase in the current carried by Kv4.3 in complex with KChIP2. These results were confirmed in mammalian CHO-K1 cells. The effect of the compound was further tested on Kv4.3/KChIP2 channels in the presence of the accessory proteins KCNE2, KCNE3, DPP6 and DPP10, which were all found to modulate the effect of NS5806. Of the various combinations of subunits investigated, we found that a complex of Kv4.3, KChIP2 and DPP6 resemble the native Ito current in canine cardiomyocytes most with regard to the effect of NS5806. The NS5806 effect on Kv4.1 and Kv4.2 was similar to the effect found for Kv4.3 both in presence and absence of KChIP2, whereas the compound had minor effect on Kv1.4 and Kv1.5 channels independent of KChIP2.Conclusion The effect of the novel Ito activator NS5806 on Kv4.3 channels was found to be strongly modulated by the presence of accessory proteins suggesting that NS5806 can be used as a valuable tool compound for addressing the molecular compositions of Ito in the heart and of IA in neuronal tissue in addition to its applicability for testing the physiological role of these currents.

Aims: The slow delayed rectifier potassium current in the heart, IKs, is important for terminating the plateau phase of the action potentials and for the repolarization of the atrial and ventricular cardiomyocytes. We set out to identify new players in cardiac repolarization that serve as regulators in the cellular network. Identification of interaction partners may allow us to understand spatial and temporal variations in ion channel function.Methods and results: We performed a yeast two-hybrid screen of a heart cDNA library using Kv7.1 N-terminus as a bait and identified the four-and-a-half LIM domain protein 2 (FHL2) as an interacting partner. We investigated the functional consequences of this interaction by expressing Kv7.1 and FHL2 in heterologous expression systems. We performed two-electrode voltage-clamp recordings on Xenopus laevis oocytes and patch-clamp experiments in mammalian cells (CHO-K1). While FHL2 did not affect the expression levels of wild-type (WT) Kv7.1 or Kv7.1/KCNE1 currents (IKs channel), it recovered two LQT5 mutants IKs channel complexes (KCNE1-D76N and KCNE1-S74L) that, typically, show markedly reduced currents in heterologous expression systems. We additionally showed that mutation in the ERK1/2 (MAPK3) phosphorylation site in Kv7.1 N-terminus removes the rescuing effect of FHL2 on the IKs-D76N mutant channel.Conclusion: With the present study, we identified two additional partners of the cardiac IKs complex that interact with Kv7.1, namely FHL2 and ERK1/2. In addition to the previously identified partners (beta-tubulin, calmodulin and Yotiao), our results show that understanding Kv7.1 intracellular regulation is important in order to comprehend the physiological effect of mutations inducing the LQT syndrome.

Changes in the lipid composition of cardiac myocytes have been reported during cardiac hypertrophy, cardiomyopathy, and infarction. Because a recent study indicates a relation between low phosphatidylinositol-bisphosphate (PIP(2)) levels and reduced intercellular coupling, we tested the hypothesis that agonist-induced changes in PIP(2) can result in a reduction of the functional coupling of cardiomyocytes and, consequently, in changes in conduction velocity. Intercellular coupling was measured by Lucifer Yellow dye transfer in cultured neonatal rat cardiomyocytes. Conduction velocity was measured in cardiomyocytes grown on microelectrode arrays. Intercellular coupling was reduced by angiotensin II (43.7 +/- 9.3%, N = 11) and noradrenaline (58.0 +/- 10.7%, N = 11). To test if reduced intercellular coupling after agonist stimulation was caused by PIP(2)-depletion, myocytes were stimulated by angiotensin II (57.3 +/- 5.7%, N = 14) and then allowed to recover in medium with or without wortmannin (an inhibitor of PIP(2) synthesis). Intercellular coupling fully recovered in control medium (102.1 +/- 8.9%, N = 10), whereas no recovery occurred in the presence of wortmannin (69.3 +/- 7.8%, N = 12). Inhibition of PKC, calmodulin, or arachidonic acid production did not affect the response to either angiotensin II or noradrenaline. Furthermore, decreasing or increasing PIP(2) also decreased and increased intercellular coupling, respectively. This supports the role of PIP(2) in the regulation of intercellular coupling. In beating myocytes, conduction velocity was reduced by angiotensin II stimulation, and recovery after wash out was prevented by inhibition of PIP(2) production. Reductions in PIP(2) inhibit intercellular coupling in cardiomyocytes, and stimulation by physiologically relevant agonists reduces intercellular coupling by this mechanism. The reduction in intercellular coupling lowered conduction velocity.

I (Kr) is the fast component of the delayed rectifier potassium currents responsible for the repolarization of the cardiac muscle. The molecular correlate underlying the I (Kr) current has been identified as the hERG1 channel. Recently, two splice variants of the hERG1 alpha-subunit, hERG1a and hERG1b, have been shown to be co-expressed in human cardiomyocytes. In this paper, we present the electrophysiological characterization of hERG1a, hERG1b, and co-expressed hERG1a/b channels in a mammalian expression system using the whole-cell patch clamp technique. We also quantified the messenger RNA (mRNA) levels of hERG1a and hERG1b in human cardiac tissue, and based on the expressed ratios, we evaluated the resulting currents in Xenopus laevis oocytes. Compared to hERG1a channels, activation was faster for both hERG1b and hERG1a/b channels. The deactivation kinetics was greatly accelerated in the presence of hERG1b, whereas no difference in the time constant of inactivation was observed. The voltage-dependent recovery from inactivation was also similar. However, the time constant of recovery from inactivation was significantly faster for hERG1b channels compared to hERG1a and hERG1a/b. Quantification of hERG1a and hERG1b mRNA in the human heart showed that hERG1b mRNA constitutes, on average, 19% in the right atrium and 12% in the left ventricle of the total hERG1 mRNA. Expression of the observed ratios of hERG1a to hERG1b in X. laevis oocytes showed that these ratios are indeed sufficient to change the deactivation phenotype markedly. The present work suggests that hERG1b is likely to play a role in the formation of the native I (Kr) current.

Voltage-gated K(+) channels of the Kv7 (KCNQ) family have important physiological functions in both excitable and nonexcitable tissue. The family encompasses five genes encoding the channel subunits Kv7.1-5. Kv7.1 is found in epithelial and cardiac tissue. Kv7.2-5 channels are predominantly neuronal channels and are important for controlling excitability. Kv7.1 channels have been considered the only Kv7 channels to undergo inactivation upon depolarization. However, here we demonstrate that inactivation is also an intrinsic property of Kv7.4 and Kv7.5 channels, which inactivate to a larger extent than Kv7.1 channels at all potentials. We demonstrate that at least 30% of these channels are inactivated at physiologically relevant potentials. The onset of inactivation is voltage dependent and occurs on the order of seconds. Both time- and voltage-dependent recovery from inactivation was investigated for Kv7.4 channels. A time constant of 1.47 +/- 0.21 s and a voltage constant of 54.9 +/- 3.4 mV were determined. It was further demonstrated that heteromeric Kv7.3/Kv7.4 channels had inactivation properties different from homomeric Kv7.4 channels. Finally, the Kv7 channel activator BMS-204352 was in contrast to retigabine found to abolish inactivation of Kv7.4. In conclusion, this work demonstrates that inactivation is a key regulatory mechanism of Kv7.4 and Kv7.5 channels.

Three members of the ERG potassium channel family have been described (ERG1-3 or Kv 11.1-3). ERG1 is by far the best characterized subtype and it constitutes the molecular component of the cardiac I(Kr) current. All three channel subtypes are expressed in neurons but their function remains unclear. The lack of functional information is at least partly due to the lack of specific pharmacological tools. The compound NS1643 has earlier been reported as an ERG1 channel activator. We found that NS1643 also activates the ERG2 channel; however, the molecular mechanism of the activation differs between the ERG1 and ERG2 channels. This is surprising since ERG1 and ERG2 channels have very similar biophysical and structural characteristics. For ERG2, NS1643 causes a left-ward shift of the activation curve, a faster time-constant of activation and a slower time-constant of inactivation as well as an increased relative importance for the fast component of deactivation to the total deactivation. In contrast, for ERG1, NS1643 causes a right-ward shift in the voltage-dependent release from inactivation but does not affect time-constants of deactivation. Because of these differences in the responses of ERG1 and ERG2 to NS1643, NS1643 can be used as a pharmacological tool to address ERG channel function. It may be useful for revealing physiological functions of ERG channels in neuronal tissue as well as to elucidate the structure-function relationships of the ERG channels.

Mutations in one of the ion channels shaping the cardiac action potential can lead to action potential prolongation. However, only in a minority of cardiac arrest cases mutations in the known arrhythmia-related genes can be identified. In two patients with arrhythmia and cardiac arrest, we identified the point mutations P91L and E33V in the KCNA5 gene encoding the Kv1.5 potassium channel that has not previously been associated with arrhythmia. We functionally characterized the mutations in HEK293 cells. The mutated channels behaved similarly to the wild-type with respect to biophysical characteristics and drug sensitivity. Both patients also carried a D85N polymorphism in KCNE1, which was neither found to influence the Kv1.5 nor the Kv7.1 channel activity. We conclude that although the two N-terminal Kv1.5 mutations did not show any apparent electrophysiological phenotype, it is possible that they may influence other cellular mechanisms responsible for proper electrical behaviour of native cardiomyocytes.

Many important physiological processes involve changes in cell volume, e.g., the transport of salt and water in epithelial cells and the contraction of muscle cells. These cells respond to swelling with a so-called regulatory volume decrease which involves the activation of K+ channels. However, the molecular identity of the involved K+ channels has not been clear, and in particular, the mechanism for activation has been obscure.

1,3-Bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) is a newly discovered activator of human ether-a-go-go-related gene (hERG) K(+) channels. Here, we characterize the effects of this compound on cloned hERG channels heterologously expressed in Xenopus laevis oocytes. When assessed with 2-s depolarizations, NS1643 enhanced the magnitude of wild-type hERG current in a concentration- and voltage-dependent manner with an EC(50) of 10.4 microM at -10 mV. The fully activated current-voltage relationship revealed that the drug increased outward but not inward currents, consistent with altered inactivation gating. NS1643 shifted the voltage dependence of inactivation by +21 mV at 10 microM and +35 mV at 30 microM, but it did not alter the voltage dependence of activation of hERG channels. The effects of the drug on three inactivation-deficient hERG mutant channels (S620T, S631A, and G628C/S631C) were determined. In the absence of channel inactivation, NS1643 did not enhance hERG current magnitude. The agonist activity of NS1643 was facilitated by mutations (F656 to Val, Met, or Thr) that are known to greatly attenuate channel inhibition by hERG blockers. We conclude that NS1643 is a partial agonist of hERG channels and that the mechanism of activation is reduced channel inactivation.

All five members of the KCNE beta-subunit family are capable of modulating the KCNQ1 potassium current. We have previously published that the murine variant of KCNE4 inhibits the human KCNQ1 current [J. Physiol. 542 (2002) 119]. Recently, this finding has been challenged by Teng et al., stating that the human variant of KCNE4 does not attenuate the KCNQ1 current but does slightly modulate the activation kinetics of the channel after expression in Xenopus laevis oocytes [Biochem. Biophys. Res. Commun. 303 (2003) 808]. In the present study, a detailed investigation on the ability of human and murine KCNE4 to affect either human or murine KCNQ1 currents has been performed. We find that the hKCNE4 subunit drastically inhibits the hKCNQ1 current after expression in X. laevis oocytes. This inhibitory effect is also observed for both hKCNE4 and mKCNE4 when either of these subunits is co-expressed with mKCNQ1. Analyses of the current properties of hKCNQ1 revealed that activation kinetics are independent of the presence of hKCNE4. hKCNE4 has, however, the ability to prevent the inactivation observed for the KCNQ1 current. Based upon previous studies and the present results, it is concluded that both hKCNE4 and mKCNE4 have a drastic inhibitory impact on both hKCNQ1 and mKCNQ1 currents.

In the heart, several K+channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K+currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K+channels, KCNQ1 and ether- a- go- go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter. Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these K+channels to the transverse tubular system close to the Ca2+channels renders them with maximal repolarizing effect.

The novel anti-ischemic compound, BMS-204352 ((3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one)), strongly activates the voltage-gated K+ channel KCNQ5 in a concentration-dependent manner with an EC50 of 2.4 microM. At 10 microM, BMS-204352 increased the steady state current at -30 mV by 12-fold, in contrast to the 2-fold increase observed for the other KCNQ channels [Schrøder et al., 2001]. Retigabine ((D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) induced a smaller, yet qualitatively similar effect on KCNQ5. Furthermore, BMS-204352 (10 microM) did not significantly shift the KCNQ5 activation curves (threshold and potential for half-activation, V1/2), as observed for the other KCNQ channels. In the presence of BMS-204352, the activation and deactivation kinetics of the KCNQ5 currents were slowed as the slow activation time constant increased up to 10-fold. The M-current blockers, linopirdine (DuP 996; 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one) and XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), inhibited the activation of the KCNQ5 channel induced by the BMS-204352. Thus, BMS-204352 appears to be an efficacious KCNQ channels activator, and the pharmacological properties of the compound on the KCNQ5 channel seems to be different from what has been obtained on the other KCNQ channels.

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.

The compound BMS-204352 has been targeted for use against acute ischemic stroke, due to its activation of the large-conductance Ca2+-activated K-channel (BK). We have previously described that the racemate (+/-)BMS-204352 reversibly modulates KCNQ4 voltage dependency. Here we show that (+/-)BMS-204352 also induces a voltage-independent KCNQ4 current. The channels were stably expressed in human embryonic kidney cells (HEK293), and investigated by use of the whole-cell mode of the patch-clamp technique. (+/-)BMS-204352 was applied extracellularly (10 microM) in order to precipitate the robust appearance of the voltage-independent current. The voltage-independent KCNQ4 currents were recorded as instantaneous increases in currents upon hyperpolarizing or depolarizing voltage steps elicited from holding potentials of -90 or -110 mV. The voltage-independent current reversed at the equilibrium potential for potassium ( E(K)), hence was carried by a K+ conductance, and was blocked by the selective KCNQ channel blockers XE991 and linopirdine. Similar results were obtained with KCNQ4 channels transiently transfected into Chinese hamster ovary cells (CHO). When (+/-)BMS-204352 was applied to stably expressed BK channels, only the voltage dependency was modulated. Retigabine, the classic activator of KCNQ channels, did not induce voltage-independent currents. Our data indicate that KCNQ4 channels may conduct voltage-dependent and voltage-independent currents in the presence of (+/-)BMS-204352.

Activation of potassium channels generally reduces cellular excitability, making potassium channel openers potential drug candidates for the treatment of diseases related to hyperexcitabilty such as epilepsy, neuropathic pain, and neurodegeneration. Two compounds, BMS-204352 and retigabine, presently in clinical trials for the treatment of stroke and epilepsy, respectively, have been proposed to exert their protective action via an activation of potassium channels. Here we show that KCNQ4 channels, stably expressed in HEK293 cells, were activated by retigabine and BMS-204352 in a reversible and concentration-dependent manner in the concentration range 0.1-10 microM. Both compounds shifted the KCNQ4 channel activation curves towards more negative potentials by about 10 mV. Further, the maximal current obtainable at large positive voltages was also increased concentration-dependently by both compounds. Finally, a pronounced slowing of the deactivation kinetics was induced in particular by BMS-204352. The M-current blocker linopirdine inhibited the baseline current, as well as the BMS-204352-induced activation of the KCNQ4 channels. KCNQ2, KCNQ2/Q3, and KCNQ3/Q4 channels were activated to a similar degree as KCNQ4 channels by 10 microM of BMS-204352 and retigabine, respectively. The compounds are, thus, likely to be general activators of M-like currents.

This study demonstrated that the methylxanthines, theophylline, IBMX and caffeine, activate the human, intermediate-conductance, Ca2+-activated K+ channel (hIK) stably expressed in HEK-293 cells. Whole-cell voltage-clamp experiments showed that the hIK current increased reversibly and voltage independently after the addition of methylxanthines. In current-clamp experiments, theophylline dose-dependently hyperpolarised the cell membrane from a resting potential of –18 mV to –56 mV. The methylxanthines did not affect large-conductance (BK) or small-conductance (SK2), Ca2+-activated K+ channels, demonstrating that the effects were not secondary to a rise in intracellular Ca2+. However, the activation of hIK by theophylline required an intracellular [Ca2+] above 30 nM. The hIK current was insensitive to 8-bromoadenosine cyclic 3′,5′-monophosphate (8-bromo-cAMP), forskolin, 8-bromoguanosine cyclic 3′,5′-monophosphate (8-bromo-cGMP) and sodium nitroprusside. Moreover, in the presence of inhibitors of protein kinase A (PKA) or protein kinase G (PKG) theophylline still activated the current. Finally, mutation of the putative PKA/PKG consensus phosphorylation site (Ser334) had no effect on the theophylline-induced activation of hIK. Since the observed activation is independent of changes in PKA/PKG-phosphorylation and of fluctuations in intracellular Ca2+, we suggest that the methylxanthines interact directly with the hIK protein.

In the heart, K+ channel-interacting protein 2 (KChIP2) stabilizes Kv4 channels in the membrane resulting in larger transient outward potassium currents (Ito). Recently it has been demonstrated that KChIP2 also enhances Cav1.2 and that the KChIP2-/- mouse has reduced inward L-type Ca2+ current. In this study, we test the impact of KChIP2-/- on cardiac contraction and on underlying Ca2+ transients.Through ultrasound examination, we found comparable left ventricular ejection fraction and fractional shortening in the heart of wild-type (WT) and KChIP2-/- mice, indicating that the function of the left ventricle was not impaired despite reduced L-type Ca2+ current. Confocal line scan Ca2+ imaging was performed using the Cal-520TM fluorophore. Whole-cell Ca2+ transients were recorded from disaggregated left ventricular cardiomyocytes at 37°C, field stimulated at cycle lengths between 80 and 1000 ms. A decrease in the relative peak of the Ca2+ transient was found in KChIP2-/- versus WT (0.58±0.06 vs. 0.71±0.07 F/F0, P

We have generated HEK293 cell lines stably expressing high levels of either the human BK channel alpha-subunit alone or the BK channel alpha-subunit and beta-subunit together. For co-expression a plasmid with three expression cassettes was constructed. Patch-clamp recordings on inside-out patches from the transfected cells resulted in macroscopic currents reflecting the expression of 200-800 BK channels per patch. No decrease in channel expression could be detected in cells grown for more than 50 passages. The alpha-subunit when expressed alone conducted currents which were sensitive to intracellular Ca2+ in the physiological range. In the presence of the beta-subunit the steady-state activation curves were shifted by -20 to -30 mV and channel deactivation kinetics were slowed. The BK channel opener NS1608 (10 microM) shifted the steady-state activation curves for the alpha-subunit as well as for the alphabeta-subunits by -40 to -50 mV.

The relaxation of rat mesenteric resistance arteries to acetylcholine was studied in vessels (normalised internal diameter 230-330 microns) mounted in an isometric myograph and contracted with noradrenaline (5 microM). Removal of the endothelium abolished acetylcholine-induced vasorelaxation, whereas pretreatment with NG-nitro-L-arginine (500 microM) only inhibited the response partly. The relaxation was, however, completely inhibited by NG-nitro-L-arginine when the arteries were contracted with 80 mM K+. Acetylcholine-induced vasorelaxation was also attenuated by pretreatment with the K+ channel blocker, iberiotoxin (100 nM), and the combined pretreatment with iberiotoxin+NG-nitro-L-arginine completely blocked vasorelaxation to acetylcholine. Further, vasorelaxation to acetylcholine was attenuated by tetraethylammonium (5 mM), 4-aminopyridine (1 mM), and BaCl2 (100 microM), respectively, whereas glibenclamide (1 microM) and indomethacin (10 microM) were devoid of effect. Vasorelaxation to the nitric oxide donor sodium nitroprusside was not influenced by iberiotoxin. We conclude that in rat mesenteric resistance arteries, there is a significant nitric oxide-independent component of acetylcholine-induced vasorelaxation, which is mediated by activation of several types of K+ channels, in particular large conductance Ca(2+)-dependent K+ channels.

The high-conductance Ca(2+)-activated K channel (BK channel) is not only regulated by a number of physiological stimuli, but it is also sensitive to pharmacological modulation. We have stably expressed the alpha-subunit of the human BK channel, hslo, in HEK 293 cells and studied by patch-clamp technique how its gating is modulated by the channel activator NS 1608, by the selective channel blocker paxilline, as well as by changes in [Ca2+]i and Vm. The cells expressed 200-800 hslo channels per patch. The channel activity was determined by tail current analysis, and the activation curves were fitted to single Boltzmann functions, from which a gating charge for the hslo channel of 1.2 elementary charges was deduced. The hslo channel was very sensitive to changes in [Ca2+]i within the physiological range, whereas Ca(2+)-independent openings were seen at Ca2+ concentrations of 15 nM or below. NS 1608 shifted the hslo channel activation curve towards negative membrane potentials with an EC50 of 2.1 microM and a maximal shift of -74 mV. The channels activated by NS 1608 were sensitive to block by paxilline, but the two molecules apparently did not interact within the same site, since paxilline reduced the size of the tail current at all voltages, whereas NS 1608 shifted the activation curve along the voltage axis. Further, the effect of paxilline was Ca(2+)-sensitive, whereas NS 1608 elicited identical effects in the presence of either < 0.5 nM or 500 nM [Ca2+]i. NS 1608 hyperpolarized the cells by -50 to -70 mV, and paxilline depolarized them towards 0 mV. In addition to the effects on the steady state current NS 1608 also significantly influenced the non-stationary channel kinetics. In the presence of NS 1608 the time constants for deactivation of tail currents were more than tripled at all potentials. We have shown, that NS 1608 modulates steady-state BK currents and channel gating kinetics through a Ca(2+)-independent interaction with the alpha-subunit of the channel.