An amino acid mutation in a potassium channel that prevents inhibition by protein kinase C.

A slowly activating, voltage-dependent potassium channel protein cloned from rat kidney was expressed in Xenopus oocytes. Two activators of protein kinase C, 1-oleoyl-2-acetyl-rac-glycerol and phorbol 12,13-didecanoate, inhibited the current. This inhibition was blocked by the kinase inhibitor staurosporine. Inhibition of the current was not seen in channels in which Ser103 was replaced by Ala, although other properties of the current were unchanged. These results indicate that inhibition of the potassium current results from direct phosphorylation of the channel subunit protein at Ser103.

Role of the ISK protein in the IminK channel complex.

The ISK (also called minK) protein, although it is structurally unrelated to any other ion channel subunit, induces slowly activating, voltage-dependent K+ channels (IminK) in Xenopus oocytes or HEK293 cells. The quaternary structure of the IminK channel complex has long remained a mystery, but recent studies suggest an interaction of the ISK protein with a traditional K+ channel subunit, identified in man as KVLQT1. It is unclear at this point what the mechanism of this interaction is, or whether the ISK protein may also interact with other ion channel subunits. However, there is an abundance of information regarding the role and regulation of the ISK protein in the IminK channel complex, discussed in this review by Andreas Busch and Hartmut Suessbrich. The ISK protein is expressed in different tissues, where IminK activation may have distinct net effects on cell function. This fact makes IminK an excellent target for pharmacological agents.

Effects of the chromanol 293B, a selective blocker of the slow, component of the delayed rectifier K+ current, on repolarization in human and guinea pig ventricular myocytes.

OBJECTIVES: The slow component of the delayed rectifier K+ current (IKs) is believed to be important in cardiac repolarization, and may be a potential target for antiarrhythmic drugs, but its study has been limited by a lack of specific blockers. The chromanol derivate 293B blocks currents expressed by minK and not HERG in Xenopus oocytes, but little is known about its effects on native currents and action potentials. We aimed to establish the effects of 293B on K+, Na+ and Ca2+ currents and action potentials in human and guinea pig cardiomyocytes. METHODS: Whole-cell patch clamp techniques were applied to assess the effects of 293B on isolated myocytes at 36 degrees C. RESULTS: Delayed rectifier current (IK) elicited by pulses to +60 mV from a holding potential of -50 mV in guinea pig myocytes was strongly inhibited by 293B (maximum inhibition 96.9 +/- 0.8%; 50% inhibitory concentration, EC50, 1.02 microM), but IK during pulses to -10 mV was unaffected (3.9 +/- 8.4% inhibition at 50 microM). Half-activation voltages, current-voltage relations, and current densities of drug-resistant and drug-sensitive IK correspond to those of IKr and IKs respectively. Inward rectifier K+ current, Na+ current and L-type Ca2+ current were unaffected by 293B. Transient outward current in human ventricular myocytes was inhibited by 293B at an EC50 of 24 microM, less than one twentieth the potency for IKs inhibition in guinea pig myocytes. While dofetilide prolonged action potential duration (APD) with strong reverse use dependence, 293B prolonged guinea pig and human ventricular APD to a similar fractional extent at all frequencies. CONCLUSIONS: 293B is a selective IKs blocker, and the frequency dependence of APD prolongation caused by this IKs blocker is different from that caused by IKr blockade: 293B may be an interesting tool to study the physiologic role of IKs and the antiarrhythmic potential of IKs blockade.

hKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant.

OBJECTIVE: The Ca(2+) independent transient outward K(+) current (I(to1)) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K(+) channel alpha-subunit contributes to the I(to1) current in many regions of the human heart. Consistently, downregulation of hKv4.3 transcripts in heart failure and atrial fibrillation is linked to reduction in I(to1) conductance. The recently cloned KChIP family of calcium sensors has been shown to modulate A-type potassium channels of the Kv4 K(+) channel subfamily. METHODS AND RESULTS: We describe the cloning and tissue distribution of hKChIP2, as well as its functional interaction with hKv4.3 after expression in Xenopus oocytes. Furthermore, we isolated a short splice variant of the hKChIP2 gene (hKCNIP2), which represents the major hKChIP2 transcript. Northern blot analyses revealed that hKChIP2 is expressed in the human heart and occurs in the adult atria and ventricles but not in the fetal heart. Upon coexpression with hKv4.3 both hKChIP2 isoforms increased the current amplitude, slowed the inactivation and increased the recovery from inactivation of hKv4.3 currents. For the first time we analyzed the influence of a KChIP protein on the voltage of half-maximal inactivation of Kv4 channels. We demonstrate that the hKChIP2 isoforms shifted the half-maximal inactivation to more positive potentials, but to a different extent. By elucidating the genomic structure, we provide important information for future analysis of the hKCNIP2 gene in candidate disorders. In the course of this work we mapped the hKCNIP2 gene to chromosome 10q24. CONCLUSIONS: Heteromeric hKv4.3/hKChIP2 currents more closely resemble native epicardial I(to1), suggesting that hKChIP2 is a true beta-subunit of human cardiac I(to1). As a result hKChIP2 might play a role in cardiac diseases, where a contribution of I(to1) has been shown.

Effects of [Ca2+]i and temperature on minK channels expressed in Xenopus oocytes.

Slowly activating, voltage-dependent minK channels cloned from rat kidney were expressed in Xenopus oocytes. Increase in the bath temperature from 22 to 32 degrees C resulted in a dramatic acceleration of minK channel activation. The extraordinarily high Q10 of minK channel activation was voltage-dependent, being higher at more negative potentials (Q10 at -20 mV; 7.02; at 20 mV: 4.0). While activation of minK channels was highly voltage-dependent at 22 degrees C, voltage had only little effect on minK channel activation at 32 degrees C. Increase in [Ca2+]i which has recently been shown to increase the maximal conductance gmax at room temperature, did not affect gmax at 32 degrees C. However, increase of [Ca2+]i caused acceleration of minK channel activation at both temperatures. The interaction of [Ca2+]i and temperature on gmax and activation rate of minK channels described here is very similar to recent findings on Ca- and temperature-effects on the slowly activating potassium conductance IKs in guinea pig cardiac myocytes.

Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole.

The widely used histamine receptor antagonists terfenadine and astemizole were shown to prolong the QT interval in electrocardiographic recordings in cases of overdose or inappropriate co-medications, indicating a possible interaction with cardiac K+ channels. Here, terfenadine and astemizole both inhibited the human ether-a-go-go related gene (HERG) encoded channels expressed in Xenopus oocytes at nanomolar concentrations in a use- and voltage-dependent fashion. In contrast, inhibition of other delayed rectifier (Kv1.1 and IsK) or inward rectifier K+ channels (IRK1) was much weaker and occurred only at high micromolar concentrations. These results suggest that blockade of HERG channels by terfenadine and astemizole might contribute to the cardiac side effects of these compounds.

Specific block of cloned Herg channels by clofilium and its tertiary analog LY97241.

The class III antiarrhythmic drug clofilium is known to block diverse delayed rectifier K+ channels at micromolar concentrations. In the present study we investigated the potency of clofilium and its tertiary analog LY97241 to inhibit K+ channels, encoded by the human ether-a-go-go related gene (HERG). Clofilium blocked HERG channels in a voltage-dependent fashion with an IC50 of 250 nM and 150 nM at 0 and +40 mV, respectively. LY97241 was almost 10-fold more potent (IC50 of 19 nM at +40 mV). Other cloned K+ channels which are also expressed in cardiac tissue, Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv4.2, Kir2.1, or I(Ks), were not affected by 100-fold higher concentrations. Block of HERG channels by LY97241 was voltage dependent and the rate of HERG inactivation was increased by LY97241. A rise of [K+]0 decreased both, rate of HERG inactivation and LY97241 affinity. The HERG S631A and S620T mutant channels which have a strongly reduced degree of inactivation were 7-fold and 33-fold less sensitive to LY97241 blockade, indicating that LY97241 binding is affected by HERG channel inactivation. In summary, the antiarrhythmic action of clofilium and its analog LY97241 appears to be caused by their potent, but distinct ability for blocking HERG channels.

Characterization of TASK-4, a novel member of the pH-sensitive, two-pore domain potassium channel family.

We report the primary sequence of TASK-4, a novel member of the acid-sensitive subfamily of tandem pore K(+) channels. TASK-4 transcripts are widely expressed in humans, with highest levels in liver, lung, pancreas, placenta, aorta and heart. In Xenopus oocytes TASK-4 generated K(+) currents displaying a marked outward rectification which was lost by elevation of extracellular K(+). TASK-4 currents were efficiently blocked by barium (83% inhibition at 2 mM), only weakly inhibited by 1 mM concentrations of quinine, bupivacaine and lidocaine, but not blocked by tetraethylammonium, 4-aminopyridine and Cs(+). TASK-4 was sensitive to extracellular pH, but in contrast to other TASK channels, pH sensitivity was shifted to more alkaline pH. Thus, TASK-4 in concert with other TASK channels might regulate cellular membrane potential over a wide range of extracellular pH.

Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1.

The polyspecific cation transporter rOCT,1 which is localized in the basolateral membrane of rat renal proximal tubules and in sinusoidal membranes of hepatocytes, was analyzed for transport of monoamine neurotransmitters. In voltage-clamp experiments with rOCT1-expressing Xenopus oocytes, superfusion with dopamine, serotonin, noradrenaline, histamine and the permanent cation acetylcholine induced saturable inwardly directed currents with apparent Km values ranging from 20 to 100 microM. Transport of dopamine was also demonstrated by uptake measurements in oocytes and in the mammalian cell line (HEK 293) which was permanently transfected with rOCT1. The high uptake rates measured in rOCT1-expressing oocytes and in transfected HEK 293 cells suggest that rOCT1 is a high capacity transporter which mediates the first step in the excretion of monoamine neurotransmitters.

Specific blockade of slowly activating I(sK) channels by chromanols -- impact on the role of I(sK) channels in epithelia.

Chromanols, which were recently shown to inhibit cAMP-mediated Cl- secretion in colon crypts via a blockade of a cAMP-activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I(sK) channels with an IC50 of 7 micromol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I(sK) inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I(sK) blocker azimilide on cAMP mediated Cl- secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl- secretion. These data suggest that I(sK) protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.

Subunit-specific inhibition of inward-rectifier K+ channels by quinidine.

Distinct inward-rectifier K+ channel subunits were expressed in Xenopus oocytes and tested for their sensitivity to the channel blocker quinidine. The 'strong' inward-rectifier K+ channel IRK1 was inhibited by quinidine with an EC50 of 0.7 mM, while the 'weak' rectifier channel ROMK1 was only moderately inhibited. ROMK1(N171D)-IRK1C-term chimeric channels, which carry both sites for strong rectification of IRK1 channels (the negatively charged D171 in the second transmembrane domain and the IRK1-C-terminus including E224), displayed strong rectification like IRK1, but showed weak sensitivity to quinidine-like ROMK1, suggesting independence of quinidine binding and rectification mechanisms. Moreover, BIR10 and BIR11, two strong rectifier subunits originally cloned from rat brain, exerted subunit-specific sensitivity to quinidine, being much higher for BIR11. Quinidine blockade of IRK1 was not voltage-dependent, but strongly dependent on the pH in the superfusate. These results strongly suggest a subunit-specific interaction of inward-rectifier K+ channels with neutral quinidine within membrane lipid bilayers.

Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors.

The suramin analogue 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X1-P2X4 as well as heteromultimeric P2X2/P2X3 receptors has been characterized. P2X1 receptors were most sensitive to inhibition by NF023 with IC50 values of 0.24 and 0.21 microM for the rat and human homologue, respectively. P2X3 receptors have an intermediate sensitivity with IC50 values of 8.5 and 28.9 microM for rat and human subtypes, respectively and P2X2 was the least sensitive subtype (IC50 > 50 microM). P2X4 receptors were insensitive to NF023 at concentrations up to 100 microM. Coexpression of rat P2X3 with rat P2X2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X3 receptors (alphabeta meATP as agonist; IC50 = 1.4 and 1.6 microM, respectively). NF023 inhibited P2X1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 microM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP (K(B) = 1.1 +/- 0.2 microM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X1 receptors heterologously expressed in Xenopus oocytes.

Synthesis and activity of novel and selective I(Ks)-channel blockers.

Since the discovery of the I(Ks)-potassium channel as the slowly activating component of the delayed rectifier current (I(k)) in cardiac tissue, the search for blockers of this current has been intense. During the screening of K(ATP)-channel openers of the chromanol type we found that chromanol 293B was able to block I(Ks). Chromanol 293B is a sulfonamide analogue of the K(ATP)-channel openers but had no activity on this target. Experiments were initiated to improve the activity and properties based on this lead compound. As a screening model we used Xenopus oocytes injected with human minK (KCNE1). Variations of the aromatic substituent and the sulfonamide group were prepared, and their activity was evaluated. We found that the greatest influence on activity was found in the aromatic substituents. The most active compounds were alkoxy substituted. We chose HMR1556 ((3R, 4S)-(+)-N-[-3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methyl-ethanesulfonamide) 10a for development as an antiarrhythmic drug. The absolute configuration, resulting from an X-ray single-crystal structure analysis, was determined.

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes.

1. The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K+ channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity. 2. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and IsK, the A-type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition < 6% at 3 microM haloperidol). 3. In contrast, haloperidol blocked HERG channels potently with an IC50 value of approximately 1 microM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC50 value of 2.6 microM. 4. Haloperidol block was use- and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested. 5. The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV. 6. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration.

1. We identified the ethacrynic-acid derivative DCPIB as a potent inhibitor of I(Cl,swell), which blocks native I(Cl,swell) of calf bovine pulmonary artery endothelial (CPAE) cells with an IC(50) of 4.1 microM. Similarly, 10 microM DCPIB almost completely inhibited the swelling-induced chloride conductance in Xenopus oocytes and in guinea-pig atrial cardiomyocytes. Block of I(Cl,swell) by DCPIB was fully reversible and voltage independent. 2. DCPIB (10 microM) showed selectivity for I(Cl,swell) and had no significant inhibitory effects on I(Cl,Ca) in CPAE cells, on chloride currents elicited by several members of the CLC-chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 microM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like I(Cl,PKA), I(Kr), I(Ks), I(K1), I(Na) and I(Ca). 3. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling-induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 microM). In agreement with the selectivity for I(Cl,swell), DCPIB did not affect atrial APD under isoosmotic conditions. 4. Preincubation of atrial cardiomyocytes with DCPIB (10 microM) completely prevented both the swelling-induced chloride currents and the AP shortening but not the hypotonic cell swelling. 5. We conclude that swelling-induced AP shortening in isolated atrial cells is mainly caused by activation of I(Cl,swell). DCPIB therefore is a valuable pharmacological tool to study the role of I(Cl,swell) in cardiac excitability under pathophysiological conditions leading to cell swelling.

A kinetic study on the stereospecific inhibition of KCNQ1 and I(Ks) by the chromanol 293B.

1. Recently we and others have demonstrated a stereoselective inhibition of slowly activating human I(Ks) (KCNQ1/MinK) and homomeric KCNQ1 potassium channels by the enantiomers of the chromanol 293B. Here, we further characterized the mechanism of the 293B block and studied the influence of the 293B enantiomers on the gating kinetics of both channels after their heterologous expression in Xenopus oocytes. 2. Kinetic analysis of currents partially blocked with 10 microM of each 293B enantiomer revealed that only 3R,4S-293B but not 3S,4R-293B exhibited a time-dependent block of I(Ks) and KCNQ1 currents, indicating preferential open channel block activity. 3. Inhibition of both KCNQ1 and I(Ks) channels by 3R,4S-293B but not by 3S,4R-293B increased during a 2 Hz train of stimuli. 4. At high extracellular potassium concentrations the inhibition of KCNQ1 by 3R,4S-293B and 3S,4R-293B was unaffected. Drug inhibition of KCNQ1 and I(Ks) by both enantiomers also did not display a significant voltage-dependence, indicating that 293B does not strongly interact with permeant ions in the pore. 5. The inhibitory properties of 3R,4S-293B on I(Ks)-channels but not those of 3S,4R-293B fulfill the theoretical requirements for a novel class III antiarrhythmic drug, i.e. positive use-dependency. This enantiomer therefore represents a valuable pharmacological tool to evaluate the therapeutic efficiency of I(Ks)blockade.

Molecular impact of MinK on the enantiospecific block of I(Ks) by chromanols.

Slowly activating I:(Ks) (KCNQ1/MinK) channels were expressed in Xenopous: oocytes and their sensitivity to chromanols was compared to homomeric KCNQ1 channels. To elucidate the contribution of the ss-subunit MinK on chromanol block, a formerly described chromanol HMR 1556 and its enantiomer S5557 were tested for enantio-specificity in blocking I:(Ks) and KCNQ1 as shown for the single enantiomers of chromanol 293B. Both enantiomers blocked homomeric KCNQ1 channels to a lesser extent than heteromeric I:(Ks) channels. Furthermore, we expressed both WT and mutant MinK subunits to examine the involvement of particular MinK protein regions in channel block by chromanols. Through a broad variety of MinK deletion and point mutants, we could not identify amino acids or regions where sensitivity was abolished or strikingly diminished (>2.5 fold). This could indicate that MinK does not directly take part in chromanol binding but acts allosterically to facilitate drug binding to the principal subunit KCNQ1.

The role of the IsK protein in the specific pharmacological properties of the IKs channel complex.

IKs channels are composed of IsK and KvLQT1 subunits and underly the slowly activating, voltage-dependent IKs conductance in heart. Although it appears clear that the IsK protein affects both the biophysical properties and regulation of IKs channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K+ channels are inhibited by the IKs blockers 293B, azimilide and 17-beta-oestradiol. However, IKs channels induced by the coexpression of IsK and KvLQT1 subunits have a 6-100 fold higher affinity for these blockers. Moreover, the IKs activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady-state currents through heteropolymeric IKs channels by arresting them in an open state. In summary, the IsK protein modulates the effects of both blockers and activators of IKs channels. This finding is important for the action and specificity of these drugs as IsK protein expression in heart and other tissues is regulated during development and by hormones.

Blockade of HERG channels by the class III antiarrhythmic azimilide: mode of action.

1. The class III antiarrhythmic azimilide has previously been shown to inhibit I(Ks) and I(Kr) in guinea-pig cardiac myocytes and I(Ks) (minK) channels expressed in Xenopus oocytes. Because HERG channels underly the conductance I(Kr), in human heart, the effects of azimilide on HERG channels expressed in Xenopus oocytes were the focus of the present study. 2. In contrast to other well characterized HERG channel blockers, azimilide blockade was reverse use-dependent, i.e., the relative block and apparent affinity of azimilide decreased with an increase in channel activation frequency. Azimilide blocked HERG channels at 0.1 and 1 Hz with IC50s of 1.4 microM and 5.2 microM respectively. 3. In an envelope of tail test, HERG channel blockade increased with increasing channel activation, indicating binding of azimilide to open channels. 4. Azimilide blockade of HERG channels expressed in Xenopus oocytes and I(Kr) in mouse AT-1 cells was decreased under conditions of high [K+]e, whereas block of slowly activating I(Ks) channels was not affected by changes in [K+]e. 5. In summary, azimilide is a blocker of cardiac delayed rectifier channels, I(Ks) and HERG. Because of the distinct effects of stimulation frequency and [K+]e on azimilide block of I(Kr) and I(Ks) channels, we conclude that the relative contribution of block of each of these cardiac delayed rectifier channels depends on heart frequency. [K+]e and regulatory status of the respective channels.

Cloning and characterization of two human polyspecific organic cation transporters.

Previously we cloned a polyspecific transporter from rat (rOCT1) that is expressed in renal proximal tubules and hepatocytes and mediates electrogenic uptake of organic cations with different molecular structures. Recently a homologous transporter from rat kidney (rOCT2) was cloned but not characterized in detail. We report cloning and characterization of two homologous transporters from man (hOCT1 and hOCT2) displaying approximately 80% amino acid identity to rOCT1 and rOCT2, respectively. Northern blots showed that hOCT1 is mainly transcribed in liver, while hOCT2 is found in kidney. Using in situ hybridization and immunohistochemistry, expression of hOCT2 was mainly detected in the distal tubule where the transporter is localized at the luminal membrane. After expression in Xenopus laevis oocytes, hOCT1 and hOCT2 mediate tracer influx of N-1-methylnicotinamide (NMN), tetraethylammonium (TEA), and 1-methyl-4-phenylpyridinium (MPP). For cation transport by hOCT2 apparent K(m) and K(i) values were determined in tracer flux measurements. In addition, electrical measurements were performed with voltage-clamped oocytes. Similar to rOCT1, cation transport by hOCT2 was pH independent, electrogenic, and polyspecific; however, the cation specificity was different. In voltage-clamped hOCT2-expressing oocytes, inward currents were induced by superfusion with MPP, TEA, choline, quinine, d-tubocurarine, pancuronium, and cyanine863. Cation transport in distal tubules is indicated for the first time. Here hOCT2 mediates the first step in cation reabsorption. hOCT1 may participate in hepatic excretion of organic cations.