Quantitative single-cell ion-channel gene expression profiling through an improved qRT-PCR technique combined with whole cell patch clamp.

Cellular excitability originates from a concerted action of different ion channels. The genomic diversity of ion channels (over 100 different genes) underlies the functional diversity of neurons in the central nervous system (CNS) and even within a specific type of neurons large differences in channel expression have been observed. Patch-clamp is a powerful technique to study the electrophysiology of excitability at the single cell level, allowing exploration of cell-to-cell variability. Only a few attempts have been made to link electrophysiological profiling to mRNA transcript levels and most suffered from experimental noise precluding conclusive quantitative correlations. Here we describe a refinement to the technique that combines patch-clamp analysis with quantitative real-time (qRT) PCR at the single cell level. Hereto the expression of a housekeeping gene was used to normalize for cell-to-cell variability in mRNA isolation and the subsequent processing steps for performing qRT-PCR. However, the mRNA yield from a single cell was insufficient for performing a valid qRT-PCR assay; this was resolved by including a RNA amplification step. The technique was validated on a stable Ltk(-) cell line expressing the Kv2.1 channel and on embryonic dorsal root ganglion (DRG) cells probing for the expression of Kv2.1. Current density and transcript quantity displayed a clear correlation when the qRT-PCR assay was done in twofold and the data normalized to the transcript level of the housekeeping gene GAPD. Without this normalization no significant correlation was obtained. This improved technique should prove very valuable for studying the molecular background of diversity in cellular excitability.

Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1.

The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.

The aromatic cluster in KCHIP1b affects Kv4 inactivation gating.

The KChIP1b splice variant has been shown to induce slow recovery from inactivation for Kv4.2 whereas KChIP1a enhanced the recovery. Both splice variants differ only by the insertion of the exon1b, rich in aromatic residues (5/11). We analysed in detail the modifications of Kv4.2 gating induced by the KChIP1b splice variant and the role for the aromatic cluster in KChIP1b in inducing these changes. By substituting alanine for the aromatic residues individually or in combination, we could convert the KChIP1b recovery behaviour into that of KChIP1a. The replacement of one or two aromatic residues resulted in a partial restitution of the KChIP1a recovery behaviour. When three aromatic residues were replaced in the exon1b, the recovery from inactivation was fast with time constants that were similar to those obtained with KChIP1a. Moreover, similar findings were observed for closed state inactivation and for the voltage dependence of inactivation. Thus, reduction of the side chain bulkiness in exon1b resulted in the conversion of the KChIP1b phenotype into the KChIP1a phenotype. These results indicate that the aromatic cluster in exon1b modulates the transitions towards and from the closed inactivated states and the steady state distribution over the respective states.

A single hERG mutation underlying a spectrum of acquired and congenital long QT syndrome phenotypes.

The long QT syndrome (LQTS) is a multi-factorial disorder that predisposes to life-threatening arrhythmias. Both hereditary and acquired subforms have been identified. Here, we present clinical and biophysical evidence that the hERG mutation c.1039 C>T (p.Pro347Ser or P347S) is responsible for both the acquired and the congenital phenotype. In one case the genotype remained silent for years until the administration of several QT-prolonging drugs resulted into a full-blown phenotype, that was reversible upon cessation of these compounds. On the other hand the mutation was responsible for a symptomatic congenital LQTS in a Dutch family, displaying a substantial heterogeneity of the clinical symptoms. Biophysical characterization of the p.Pro347Ser potassium channels using whole-cell patch clamp experiments revealed a novel pathogenic mechanism of reciprocal changes in the inactivation kinetics combined with a dominant-negative reduction of the functional expression in the heterozygous situation, yielding a modest genetic predisposition for LQTS. Our data show that in the context of the multi-factorial aetiology underlying LQTS a modest reduction of the repolarizing power can give rise to a spectrum of phenotypes originating from one mutation. This observation increases the complexity of genotype-phenotype correlations in more lenient manifestations of the disease and underscores the difficulty of predicting the expressivity of the LQTS especially for mutations with a more subtle impact such as p.Pro347Ser.

Modulation of HERG gating by a charge cluster in the N-terminal proximal domain.

Human ether-a-go-go-related gene (HERG) potassium channels contribute to the repolarization of the cardiac action potential and display unique gating properties with slow activation and fast inactivation kinetics. Deletions in the N-terminal 'proximal' domain (residues 135-366) have been shown to induce hyperpolarizing shifts in the voltage dependence of activation, suggesting that it modulates activation. However, we did not observe a hyperpolarizing shift with a subtotal deletion designed to preserve the local charge distribution, and other deletions narrowed the region to the KIKER containing sequence 362-372. Replacing the positively charged residues of this sequence by negative ones (EIEEE) resulted in a -45 mV shift of the voltage dependence of activation. The shifts were intermediate for individual charge reversals, whereas E365R resulted in a positive shift. Furthermore, the shifts in the voltage dependence were strongly correlated with the net charge of the KIKER region. The apparent speeding of the activation was attributable to the shifted voltage dependence of activation. Additionally, the introduction of negative charges accelerated the intermediate voltage-independent forward rate constant. We propose that the modulatory effects of the proximal domain on HERG gating are largely electrostatic, localized to the charged KIKER sequence.

Obligatory heterotetramerization of three previously uncharacterized Kv channel alpha-subunits identified in the human genome.

Voltage-gated K(+) channels control excitability in neuronal and various other tissues. We identified three unique alpha-subunits of voltage-gated K(+)-channels in the human genome. Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1. Although they have all of the hallmarks of voltage-gated K(+) channel subunits, they did not produce K(+) currents when expressed in mammalian cells. Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum. Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1. Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents. This heteromerization was confirmed by co-immunoprecipitation. The Kv2 subfamily consists of only two members and uses interaction with "silent subunits" to diversify its function. Including the subunits described here, the "silent subunits" represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.

Drug block of I(kr): model systems and relevance to human arrhythmias.

The long QT-related arrhythmia torsades de pointes (TdP) can arise with mutations in HERG and during treatment with drugs that block cardiac I Kr, the current encoded by HERG. Multiple test systems have been used to assess drug block of I Kr. This study evaluated the I Kr blocking potency of a series of antiarrhythmics associated with a range of clinical risks of TdP in two such systems: mouse AT-1 cells (in which I Kr is the major repolarizing current) and Ltk cells transiently transfected with HERG (n = 4-10 cells per drug). For each compound, the concentration required to produce 50% block of I Kr or HERG tail currents (IC 50 ) was determined. There was an excellent correlation ( r = 0.98, p < 10 -5 ) between values obtained in the two systems. However, the relation between the liability of a drug to cause TdP appeared dissociated from I Kr blocking potency. Quinidine, dofetilide, ibutilide, procainamide, and disopyramide are all associated with TdP, but only the first three were potent blockers (IC 50 < or = 1 microM ), whereas procainamide and disopyramide were not (IC 50 > 50 microM ). Conversely, verapamil and amiodarone, drugs not associated with TdP, were also blockers (IC 50 < or = 1 microM ). We conclude that I Kr blocking potency can be readily assessed in either AT-1 cells or systems in which HERG is heterologously expressed. However, not all drugs causing TdP are potent I Kr blockers, and I Kr block is not necessarily associated with TdP. Other properties of these drugs, therefore, contribute to their propensity to cause TdP.

Effects of antiarrhythmic drugs on cloned cardiac voltage-gated potassium channels expressed in Xenopus oocytes.

The effects of 17 commonly used antiarrhythmic drugs on the rapidly activating cardiac voltage-gated potassium channels (Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1 and Kv4.2) were studied in the expression system of the Xenopus oocyte. A systematic overview on basic properties was obtained using a simple and restricted experimental protocol (command potentials 10 mV and 50 mV positive to the threshold potential; concentration of 100 micromol/l each). The study revealed that 8 of 17 drugs yielded significant effects (changes >10% of control) on at least one type of potassium channel in the oocyte expression system. These drugs were ajmaline, diltiazem, flecainide, phenytoin, propafenone, propranolol, quinidine and verapamil, whereas the effects of adenosine, amiodarone, bretylium, disopyramide, lidocaine, mexiletine, procainamide, sotalol and tocainide were negligible. The drug effects were characterized by reductions of the potassium currents (except for quinidine and ajmaline). A voltage-dependence of drug effect was found for quinidine, verapamil and diltiazem. The different effect of the drugs was not related to the fast or slow current inactivation of the potassium channels (except for verapamil). Profiles of the individual drug effects at the different potassium channel types were identical for propafenone and flecainide and differed for all other substances. The study demonstrates marked differences in sensitivity to antiarrhythmic drugs within the group of voltage-operated cardiac potassium channel types. Taking the restrictions of the oocyte system into consideration, the findings suggest that several antiarrhythmic drugs exert significant effects at rapidly activating cardiac potassium channels.

Effects of a quaternary bupivacaine derivative on delayed rectifier K(+) currents.

Block of hKv1.5 channels by R-bupivacaine has been attributed to the interaction of the charged form of the drug with an intracellular receptor. However, bupivacaine is present as a mixture of neutral and charged forms both extra- and intracellularly. We have studied the effects produced by the R(+) enantiomer of a quaternary bupivacaine derivative, N-methyl-bupivacaine, (RB(+)1C) on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell configuration of the patch-clamp technique. When applied from the intracellular side of the membrane, RB(+)1C induced a time- and voltage-dependent block similar to that induced by R-bupivacaine. External application of 50 microM RB(+)1C reduced the current at +60 mV by 24+/-2% (n=10), but this block displayed neither time- nor voltage-dependence. External RB(+)1C partially relieved block induced by R-bupivacaine (61+/-2% vs 56+/-3%, n=4, P<0.05), but it did not relieve block induced by internal RB(+)1C. In addition, it did not induce use-dependent block, but when applied in combination with internal RB(+)1C a use-dependent block that increased with pulse duration was observed. These results indicate that RB(+)1C induces different effects on hKv1.5 channels when applied from the intra or the extracellular side of the membrane, suggesting that the actions of bupivacaine are the resulting of those induced on the external and the internal side of hKv1.5 channels.

Structure and function of cardiac potassium channels.

Recent advances in molecular biology have had a major impact on our understanding of the biophysical and molecular properties of ion channels. This review is focused on cardiac potassium channels which, in general, serve to control and limit cardiac excitability. Approximately 60 K+ channel subunits have been cloned to date. The (evolutionary) oldest potassium channel subunits consist of two transmembrane (Tm) segments with an intervening pore-loop (P). Channels formed by four 2Tm-1P subunits generally function as inwardly rectifying K(+)-selective channels (KirX.Y): they conduct substantial current near the resting potential but carry little or no current at depolarized potentials. The inward rectifier IK1 and the ligand-gated KATP and KACh channels are composed of such subunits. The second major class of K+ channel subunits consists of six transmembrane segments (S1-S6). The S5-P-S6 section resembles the 2Tm-1P subunit, and the additional membrane-spanning segments (especially the charged S4 segment) endow these 6Tm-1P channels with voltage-dependent gating. For both major families, four subunits assemble into a homo- or heterotetrameric channel, subject to specific subunit-subunit interactions. The 6Tm-1P channels are closed at the resting potential, but activate at different rates upon depolarization to carry sustained or transient outward currents (the latter due to inactivation by different mechanisms). Cardiac cells typically display at least one transient outward current and several delayed rectifiers to control the duration of the action potential. The molecular basis for each of these currents is formed by subunits that belong to different Kvx.y subfamilies and alternative splicing can contribute further to the diversity in native cells. These subunits display distinct pharmacological properties and drug-binding sites have been identified. Additional subunits have evolved by concatenation of two 2Tm-1P subunits (4Tm-2P); dimers of such subunits yield voltage-independent leak channels. A special class of 6Tm-1P subunits encodes the 'funny' pacemaker current which activates upon hyperpolarization and carries both Na+ and K+ ions. The regional heterogeneity of K+ currents and action potential duration is explained by the heterogeneity of subunit expression, and significant changes in expression occur in cardiac disease, most frequently a reduction. This electrical remodelling may also be important for novel antiarrhythmic therapeutic strategies. The recent crystallization of a 2Tm-1P channel enhances the outlook for more refined molecular approaches.

Benzocaine enhances and inhibits the K+ current through a human cardiac cloned channel (Kv1.5).

OBJECTIVE: The aim of this study was to analyze the effects of a neutral local anaesthetic, benzocaine, on a cardiac K+ channel cloned from human ventricle. METHODS: Experiments were performed on hKv1.5 channels stably expressed on mouse cells using the whole-cell configuration of the patch clamp technique. RESULTS: At 10 nM, benzocaine increased the current amplitude ("agonist effect") by shifting the activation curve 8.4 +/- 2.7 mV in the negative direction, and slowed the time course of tail current decline. In contrast, benzocaine (100-700 microM) inhibited hKv1.5 currents (KD = 901 +/- 81 microM), modified the voltage-dependence of channel activation, which became biphasic, and accelerated the channel deactivation. Extracellular K+ concentration ([K+]o) also affected the channel gating. At 140 mM [K+]o, the time course of tail currents deactivation was significantly accelerated, whereas at 0 mM [K+]o, it was slowed. At both [K+]o the activation curve became biphasic. Benzocaine accelerated the tail current decay at 0 mM but not at 140 mM [K+]o. The reduction in the permeation of K+ through the pore did not modify the blocking effects of micromolar concentrations of benzocaine, but suppressed the agonist effect observed at nanomolar concentrations. CONCLUSIONS: All these results suggest that benzocaine blocks and modifies the voltage- and time-dependent properties of hKv1.5 channels, binding to an extracellular and to an intracellular site at the channel level. Moreover, both sites are related to each other and can also interact with K+.

Functional expression of an inactivating potassium channel (Kv4.3) in a mammalian cell line.

OBJECTIVE: The goal of this study was to characterize the electrophysiological properties of the Kv4.3 channels expressed in a mammalian cell line. METHODS: Currents were recorded using the whole-cell voltage clamp technique. RESULTS: The threshold for activation of the expressed Kv4.3 current was approximately -30 mV. The dominant time constant for activation was 1.71 +/- 0.16 ms (n = 10) at +60 mV. The current inactivated, this process being incomplete, resulting in a sustained level which contributed 15 +/- 2% (n = 25) of the total current. The time course of inactivation was fit by a biexponential function, the fast component contributing 74 +/- 5% (n = 9) to the overall inactivation. The fast time constant was voltage-dependent [27.6 +/- 2.0 ms at +60 mV (n = 10) versus 64.0 +/- 3.6 ms at 0 mV (n = 10); P < 0.01], whereas the slow was voltage-independent [142 +/- 15 ms at +60 mV (n = 10) versus 129 +/- 33 ms at 0 mV (n = 6) P > 0.05]. The voltage-dependence of inactivation exhibited midpoint and slope values of -26.9 +/- 1.5 mV and 5.9 +/- 0.3 mV (n = 21). Recovery from inactivation was faster at more negative membrane potentials [203 +/- 17 ms (n = 13) and 170 +/- 19 ms (n = 4), at -90 and -100 mV]. Bupivacaine block of Kv4.3 channels was not stereoselective (KD approximately 31 microM). CONCLUSIONS: The functional profile of Kv4.3 channels expressed in Ltk- cells corresponds closely to rat ITO, although differences in recovery do not rule out association with accessory subunits. Nevertheless, the sustained component needs to be considered with respect to native ITO.

A K+ channel splice variant common in human heart lacks a C-terminal domain required for expression of rapidly activating delayed rectifier current.

We have cloned HERG USO, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (IKr), from human heart, and we find that its mRNA is approximately 2-fold more abundant than that for HERG1 (the originally described cDNA). After transfection of HERG USO in Ltk- cells, no current was observed. However, coexpression of HERG USO with HERG1 modified IKr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERG USO, HERGDeltaC (a HERG1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, IKr was rescued by ligation of 104 amino acids from the C terminus of HERG1 to the C terminus of HERGDeltaC, indicating that the C terminus of HERG1 includes a domain (

Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier.

The kinetic properties of hKv1.5, a Shaker-related cardiac delayed rectifier, expressed in Ltk- cells were studied. hKv1.5 currents elicited by membrane depolarizations exhibited a delay followed by biphasic activation. The biphasic activation remained after 5-s prepulses to membrane potentials between -80 and -30 mV; however, the relative amplitude of the slow component increased as the prepulse potential approached the threshold of channel activation, suggesting that the second component did not reflect activation from a hesitant state. The decay of tail currents at potentials between -80 and -30 mV was adequately described with a biexponential. The time course of deactivation slowed as the duration of the depolarizing pulse increased. This was due to a relative increase in the slowly decaying component, despite similar initial amplitudes reflecting a similar open probability after 50- and 500-ms prepulses. To further investigate transitions after the initial activated state, we examined the temperature dependence of inactivation. The time constants of slow inactivation displayed little temperature and voltage dependence, but the degree of the inactivation increased substantially with increased temperature. Recovery from inactivation proceeded with a biexponential time course, but long prepulses at depolarized potentials slowed the apparent rate of recovery from inactivation. These data strongly indicate that hKv1.5 has both multiple open states and multiple inactivated states.

Distinct domains of the voltage-gated K+ channel Kv beta 1.3 beta-subunit affect voltage-dependent gating.

The Kvbeta1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 +/- 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta1.3-mediated effects involve at least two distinct domains of this beta-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels.

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Block of human cardiac Kv1.5 channels by loratadine: voltage-, time- and use-dependent block at concentrations above therapeutic levels.

OBJECTIVE: The aim of this study was to analyze the effects of loratadine on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line. METHODS: Currents were studied using the whole-cell configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding hKv1.5 channels. RESULTS: Loratadine inhibited in a concentration-dependent manner the hKv1.5 current, the apparent affinity being 1.2 +/- 0.2 microM. The blockade increased steeply between -30 and 0 mV which corresponded with the voltage range for channel opening, thus suggesting that the drug binds preferentially to the open state of the channel. The apparent association and dissociation rate constants were (3.6 +/- 0.5) x 10(6).M-1.s-1 and 3.7 +/- 1.6.s-1, respectively. Loratadine, 1 microM, increased the time constant of deactivation of tail currents elicited on return to -40 mV after 500 ms depolarizing pulses to +60 mV from 36.2 +/- 3.4 to 64.9 +/- 3.6 ms (n = 6, P < 0.01), thus inducing a 'crossover' phenomenon. Application of trains of pulses at 1 Hz lead to a progressive increase in the blockade reaching a final value of 48.6 +/- 4.3%. Recovery from loratadine-induced block at -80 mV exhibited a time constant of 743.0 +/- 78.0 ms. Finally, the results of a mathematical stimulation of the effects of loratadine, based on an open-channel block model, reproduced fairly well the main effects of the drug. CONCLUSIONS: The present results demonstrated that loratadine blocked hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner but only at concentrations much higher than therapeutic plasma levels in man.

Rapid inactivation determines the rectification and [K+]o dependence of the rapid component of the delayed rectifier K+ current in cardiac cells.

Two characteristic features of the rapid component of the cardiac delayed rectifier current (IKr) are prominent inward rectification and an unexpected reduction in activating current with decreased [K+]o. Similar features are observed with heterologous expression of HERG, the gene thought to encode the channel carrying IKr, moreover, recent studies indicate that the mechanism underlying rectification of HERG current is the inactivation that channels rapidly undergo during depolarizing pulses. The present studies were designed to determine the mechanism of IKr rectification and [K+]o sensitivity in the mouse atrial myocyte cell line, AT-1 cells. Reducing [Mg2+]i to 0, which reverses inward rectification of some K+ channels, did not alter IKr current-voltage relationships, although it did decrease sensitivity to the IKr blockers dofetilide and quinidine 2- to 5-fold. To determine the presence and extent of fast inactivation of IKr in AT-1 cells, a brief hyperpolarizing pulse (20 ms to -120 mV) was applied during long depolarizations. Immediately after this pulse, a very large outward current that decayed rapidly to the previous activating current baseline was observed. This outward current component was blocked by the IKr-specific inhibitor dofetilide, indicating that it represented recovery from fast inactivation during the hyperpolarizing step, with fast reinactivation during the return to depolarized potential. With removal of inactivation using this approach, current-voltage relationships for IKr ([K+]o, 1 to 20 mmol/L) were linar and reversed close to the predicted Nernst potential for K+. In addition, decreased [K+]o decreased the time constants for open-->inactivated and inactivated-->open transitions. Thus, in these cardiac myocytes, as with heterologously expressed HERG, IKr undergoes fast inactivation that determines its characteristic inward rectification. These studies demonstrate that the mechanism underlying decreased activating current observed at low [K+]o is more extensive fast inactivation.

Electrophysiological and pharmacological correspondence between Kv4.2 current and rat cardiac transient outward current.

OBJECTIVE: The transient outward current (ITO) plays an important role in early repolarization and overall time course of the cardiac action potential. At least two K+ channel alpha-subunits cloned from cardiac tissue (Kv1.4 and Kv4.2) encode rapidly inactivating channels. The goal of this study was to determine functional and pharmacological properties of Kv4.2 expressed in mammalian cells, especially those that would differentiate between both isoforms in comparison to native ITO. METHODS: Both Kv4.2 and Kv1.4 isoforms were stably expressed in mouse L-cell lines, and expressed currents were studied using whole-cell voltage clamp techniques. RESULTS: The expressed Kv4.2 currents displayed fast inactivation with a half-inactivation potential of -41 mV. Recovery from inactivation was rapid (tau recov = 160 ms at -90 mV) and strongly voltage-dependent. Flecainide (10 microM) had minimal effects on Kv1.4 currents, but reduced Kv4.2 peak current by 53% and increased the apparent rate of inactivation consistent with open channel block. Quinidine (10-20 microM) reduced the peak current and accelerated the apparent rate of inactivation in both isoforms. The Kv4.2 current displayed use-dependent unblock in the presence of 4-AP. CONCLUSIONS: The functional properties of Kv4.2, especially the flecainide sensitivity, resemble those of ITO in rat (and human) myocytes better than those of Kv1.4. These results provide the necessary functional support for the hypothesis that Kv4.2 is a major isoform contributing to cardiac ITO, consistent with independent biochemical and molecular evidence that indicates that Kv4.2 is readily detected in rat myocytes.

Inhibition of cardiac potassium currents by the vesnarinone analog OPC-18790: comparison with quinidine and dofetilide.

OPC-18790 is a vesnarinone analog currently in clinical trials for treatment of heart failure. In vitro studies have shown that, in addition to its positive inotropic actions, OPC-18790 prolongs cardiac action potentials. Therefore, in this study, the effects of OPC-18790 on cardiac potassium currents were compared with those we previously observed for the blockers quinidine and dofetilide in two test systems, i.e., L-cells stably transfected with mammalian cardiac potassium channel clones (Kv1.4, Kv1.5 and Kv2.1) and mouse AT-1 cells, in which the rapidly inactivating component of the cardiac delayed rectifier (I(Kr)) is the major repolarizing current. In L-cells, 10 to 100 microM OPC-18790 reduced Kv1.4, Kv1.5 and Kv2.1 currents by <30%, whereas quinidine was a more potent blocker (EC50 < 10 microM) and the I(Kr)-specific blocker dofetilide was without effect. In contrast, in AT-1 cells, OPC-18790 blocked I(Kr) with an EC50 (0.96 +/- 0.12 microM, n = 10) similar to that of quinidine (0.9 +/- 0.2 microM). For both drugs, block was voltage dependent, increasing at positive potentials. OPC-18790 and quinidine showed no frequency dependence, implying block of resting channels and/or very rapid block of open channels; this is in contrast to dofetilide, which displayed slow onset kinetics of block. Thus, we conclude that, 1) unlike quinidine, OPC-18790 does not significantly inhibit currents obtained by expression of the cardiac potassium channel clones Kv1.4, Kv1.5 and Kv2.1; 2) like quinidine and dofetilide, OPC-18790 blocks I(Kr) in AT-1 cells, but the kinetics of block onset more closely resemble those of quinidine than dofetilide; and 3) block of I(Kr) appears to be an important mechanism underlying the action potential-prolonging properties of OPC-18790.