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The in vitro cardiac properties of dihydroartemisinin (DHA) plus piperaquine phosphate (PQP) were compared with those of other antimalarial compounds. Results with antimalarial drugs, chosen on the basis of their free therapeutic maximum concentration in plasma (C(max)), were expressed as the fold of that particular effect with respect to their C(max). The following tests were used at 37 °C: hERG (human ether-à-go-go-related gene) blockade and trafficking, rabbit heart ventricular preparations, and sodium and slow potassium ion current interference (I(Na) and I(Ks), respectively). Chloroquine, halofantrine, mefloquine, and lumefantrine were tested in the hERG studies, but only chloroquine, dofetilide, lumefantrine, and the combination of artemether-lumefantrine were used in the rabbit heart ventricular preparations, hERG trafficking studies, and I(Na) and I(Ks) analyses. A proper reference was used in each test. In hERG studies, the high 50% inhibitory concentration (IC(50)) of halofantrine, which was lower than its C(max), was confirmed. All the other compounds blocked hERG, with IC(50)s ranging from 3- to 30-fold their C(max)s. In hERG trafficking studies, the facilitative effects of chloroquine at about 30-fold its C(max) were confirmed and DHA blocked it at a concentration about 300-fold its C(max). In rabbit heart ventricular preparations, dofetilide, used as a positive control, revealed a high risk of torsades de pointes, whereas chloroquine showed a medium risk. Neither DHA-PQP nor artemether-lumefantrine displayed an in vitro signal for a significant proarrhythmic risk. Only chloroquine blocked the I(Na) ion current and did so at about 30-fold its C(max). No effect on I(Ks) was detected. In conclusion, despite significant hERG blockade, DHA-PQP and artemether-lumefantrine do not appear to induce potential torsadogenic effects in vitro, affect hERG trafficking, or block sodium and slow potassium ion currents.

Spatial heterogeneity of the action potential and its influence on arrhythmia vulnerability is known. However, heterogeneity of intracellular calcium handling and, in particular, its effect on the electrophysiological substrate is less clear. Using optical mapping techniques, calcium transients and action potentials were recorded simultaneously from ventricular sites across the transmural wall of the arterially perfused canine left ventricular wedge preparation during steady-state baseline pacing and rapid pacing. During baseline pacing, the decay of intracellular calcium to diastolic levels and calcium transient duration were slower (70%, P<0.005) and longer (20%, P<0.005), respectively, closer to the endocardial surface compared with the epicardial surface. Tissue samples isolated from the left ventricular wall demonstrate that sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression was significantly less in the subendocardial and midmyocardial layers compared with the subepicardial layer. In contrast, no significant difference in the transmural expression of Na+-Ca2+ exchanger was observed. During rapid pacing, calcium transient alternans and increased levels of diastolic intracellular calcium were significantly greater (P<0.01) closer to the endocardium (101%+/-62% and 41%+/-15%, respectively) compared with the epicardium (12%+/-7% and 12%+/-14%, respectively). In conclusion, cells closer to the endocardium exhibit a slower decay of intracellular calcium compared with cells near the epicardium, which may be due in part to reduced expression of SERCA2a. As a possible consequence, calcium transient alternans and increased diastolic levels of intracellular calcium may occur preferentially closer to the endocardial surface.

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.

Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme (). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kv beta 2 with Kv alpha 1 channels. To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kv beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kv beta 2 may play a role in the processing of Kv1.4.

Hereditary long QT syndrome (hLQTS) is a heterogeneous genetic disease characterized by prolonged QT interval in the electrocardiogram, recurrent syncope, and sudden cardiac death. Mutations in the cardiac potassium channel HERG (KCNH2) are the second most common form of hLQTS and reduce the delayed rectifier K(+) currents, thereby prolonging repolarization. We studied a novel COOH-terminal missense mutation, HERG R752W, which segregated with the disease in a family of 101 genotyped individuals. When the mutant cRNA was expressed in Xenopus oocytes it produced enhanced rather than reduced currents. Simulations using the Luo-Rudy model predicted minimal shortening rather than prolongation of the cardiac action potential. Consequently, a normal or shortened QT interval would be expected in contrast to the long QT observed clinically. This anomaly was resolved by our observation that the mutant protein was not delivered to the plasma membrane of mammalian cells but was retained intracellularly. We found that this trafficking defect was corrected at lower incubation temperatures and that functional channels were now delivered to the plasma membrane. However, trafficking could not be restored by chemical chaperones or E-4031, a specific blocker of HERG channels. Therefore, HERG R752W represents a new class of trafficking mutants in hLQTS. The occurrence of different classes of misprocessed channels suggests that a unified therapeutic approach for altering HERG trafficking will not be possible and that different treatment modalities will have to be matched to the different classes of trafficking mutants.

The concept of chaperones for K(+) channels is new. Recently, we discovered a novel molecular chaperone, KChAP, which increased total Kv2.1 protein and functional channels in Xenopus oocytes through a transient interaction with the Kv2.1 amino terminus. Here we report that KChAP is a chaperone for Kv1.3 and Kv4.3. KChAP increased the amplitude of Kv1.3 and Kv4.3 currents without affecting kinetics or voltage dependence, but had no such effect on Kv1.1, 1.2, 1.4, 1.5, 1.6, and 3.1 or Kir2.2, HERG, or KvLQT1. Although KChAP belongs to a family of proteins that interact with transcription factors, upregulation of channel currents was not blocked by the transcription inhibitor actinomycin D. A 98-amino acid fragment of KChAP binds to the channel and is indistinguishable from KChAP in its enhancement of Kv4.3 current and protein levels. Using a KChAP antibody, we have coimmunoprecipitated KChAP with Kv2.1 and Kv4.3 from heart. We propose that KChAP is a chaperone for specific Kv channels and may have this function in cardiomyocytes where Kv4.3 produces the transient outward current, I(to).

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) that plays a fundamental role in heart excitability by regulating the action potential repolarization (IKr); mutations of this gene are responsible for the chromosome 7-linked long QT syndrome (LQT2). In this report, we show that in a variety (n = 17) of tumor cell lines of different species (human and murine) and distinct histogenesis (neuroblastoma, rhabdomyosarcoma, adenocarcinoma, lung microcytoma, pituitary tumors, insulinoma beta-cells, and monoblastic leukemia), a novel K+ inward-rectifier current (IIR), which is biophysically and pharmacologically similar to IHERG, can be recorded with the patch-clamp technique. Northern blot experiments with a human herg cDNA probe revealed that both in human and murine clones the very high expression of herg transcripts can be quantified in at least three clearly identifiable bands, suggesting an alternative splicing of HERG mRNA. Moreover, we cloned a cDNA encoding for IIR from the SH-SY5Y human neuroblastoma. The sequence of this cDNA result was practically identical to that already reported for herg, indicating a high conservation of this gene in tumors. Consistently, the expression of this clone in Xenopus oocytes showed that the encoded K+ channel had substantially all of the biophysical and pharmacological properties of the native IIR described for tumor cells. In addition, in the tumor clones studied, IIR governs the resting potential, whereas it could not be detected either by the patch clamp or the Northern blot techniques in cells obtained from primary cell cultures of parental tissues (sensory neurons and myotubes), whose resting potential is controlled by the classical K+ anomalous rectifier current. This current substitution had a profound impact on the resting potential, which was markedly depolarized in tumors as compared with normal cells. These results suggest that IIR is normally only expressed during the early stages of cell differentiation frozen by neoplastic transformation, playing an important pathophysiological role in the regulatory mechanisms of neoplastic cell survival. In fact, because of its biophysical features, IIR, besides keeping the resting potential within the depolarized values required for unlimited tumor growth, could also appear suitable to afford a selective advantage in an ischemic environment.

Experiments were carried out to determine whether coinjection of Kvalpha1.2 with inactivating and noninactivating Kvbeta subunits would produce currents with intermediate kinetics and channel complexes containing a mixture of these subunits. Upon coexpression with a saturating amount of Kvbeta1.2 and increasing levels of a noninactivating deletion mutant of Kvbeta1.2, we show that macroscopic Kvalpha1.2 currents have levels of fractional inactivation and inactivation time constants that are intermediate between those obtained with either the inactivating Kvbeta1.2 or the noninactivating Kvbeta1.2 mutant. We also find that coexpression of Kvalpha1.2 with saturating amounts of Kvbeta1.2 and the deletion mutant produces a population of single channels with properties intermediate to either the inactivating or noninactivating parental phenotype. Our data can best be explained by the presence of an intermediate population of heterooligomeric channels consisting of Kvalpha1.2 with different combinations of both types of subunits. Since Kvalpha1.2 subunits coexist in cells with inactivating and noninactivating Kvbeta subunits, our findings suggest that heterooligomeric assembly of these subunits occurs to increase the range of K+ current kinetics and expression levels.

Several cloned K+ channel subunits are candidates to underlie macroscopic currents in the human heart, but direct evidence bearing on their role is lacking. The Kv1.5 K+ channel subunit has been suggested to play a potential role in human cardiac ultrarapid delayed rectifier (IKur) and transient outward (Ito) currents. To evaluate the role of proteins encoded by the Kv1.5 gene, we incubated cultured human atrial myocytes for 48 hours in medium containing antisense phosphorothioate oligodeoxynucleotides directed against octodecameric segments of the Kv1.5 mRNA coding sequence, the same concentration of homologous oligodeoxynucleotides with four mismatch mutations, or vehicle (control group). Cells exposed to antisense showed a highly significant (approximately 50%) reduction in IKur whether measured by step current at the end of a 400-millisecond depolarizing pulse, tail current at -20 mV, or current sensitive to a concentration of 4-aminopyridine (50 mumol/L) that is highly selective for IKur compared with control cells or cells exposed to mismatch oligodeoxynucleotides. In contrast, Ito was not different among the three experimental groups. When cultured human ventricular myocytes were exposed to Kv1.5 antisense oligodeoxynucleotides with the same controls, no changes occurred in either Ito or the sustained current at the end of a depolarizing pulse. We conclude that Kv1.5 channel subunits are essential to the expression of IKur and do not play a role in Ito in cultured human atrial myocytes. These studies provide the first direct evidence with an antisense approach for the equivalence between a macroscopic cardiac K+ current and a cloned K+ channel subunit and offer insights into the molecular electrophysiology of the human heart.

Background: The human ether-a-go-go-related gene (HERG) is one locus for the hereditary long-QT syndrome. A hypothesis is that HERG produces the repolarizing cardiac potassium current IKr with the consequence that mutations in HERG prolong the QT interval by reducing IKr. The elementary properties of HERG are unknown, and as a test of the hypothesis that HERG produces IKr, we compared their elementary properties., Methods and Results: We injected HERG cRNA into Xenopus oocytes and measured currents from single channels or current variance from the noise produced by ensembles of channels recorded from macro patches. Single-channel conductance was dependent on the extracellular potassium concentration ([K]o). At physiological [K]o, it was 2 picosiemens (pS), and at 100 mmol/L [K]o, it was 10 pS. Openings occurred in bursts with a mean duration of 26 ms at -100 mV. Mean open time was 3.2 ms and closed times were 1.0 and 26 ms. In excised macro patches, HERG currents were blocked by the class III antiarrhythmic drug dofetilide, with an IC50 of 35 nmol/L. Dofetilide block was slow and greatly attenuated at positive potentials at which HERG rectifies., Conclusions: The microscopic physiology of HERG and IKr is similar, consistent with HERG being an important component of IKr. The pharmacology is also similar; dofetilide appears to primarily block activated channels and has a much lower affinity for closed and inactivated channels.

Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as IKr, the rapidly activating component of the repolarizing outward cardiac K+ current, IK. IKr may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K+ (Kv) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K+ current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K+ rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in Xenopus oocytes, are blocked by dofetilide at submicromolar concentrations (IC50 = 533 nmol/L at 40 mV and 20 degrees C). The drug has no significant blocking effect on the human cardiac Kv channels hKv1.2, hKv1.4, hKv1.5, or hKv2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau = 1.17 seconds at -80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.

We report the cloning and functional expression of a novel K+ channel beta-subunit from human atrium, hKv beta 3. hKv beta 3 is highly homologous to the two beta-subunits cloned from rat brain, Kv beta 1 and Kv beta 2, but has an essentially unique stretch of 79 N-terminal residues. Upon expression in Xenopus oocytes, hKv beta 3 accelerates the inactivation of co-injected hKv1.4 currents and induces fast inactivation of non-inactivating co-injected hKv1.5 currents. By contrast, hKv beta 3 had no effect on hKv1.1, hKv1.2, or hKv2.1 currents. Thus, hKv beta 3 represents a third type of K+ channel beta-subunit which modulates the kinetics of a unique subset of channels in the Kv1 subfamily.

The cardiac inward rectifier current (IK1) contributes to the shape and duration of the cardiac action potential and helps to set the resting membrane potential. Although several inwardly rectifying K+ channels (IRKs) from different tissues have been cloned recently, the nature and number of K+ channels contributing to the cardiac IK1 are presently unknown. To address this issue in human heart, we have used the reverse-transcriptase-polymerase chain reaction (PCR) technique with human atrial total RNA as a template to identify two sequences expressed in heart that are homologous to previously cloned IRKs. One of the PCR products we obtained was virtually identical to IRK1 (cloned from a mouse macrophage cell line); the other, which we named hIRK, exhibited < 70% identity to IRK1. A full-length clone encoding hIRK was isolated from a human atrial cDNA library and functionally expressed in Xenopus oocytes. This channel, like IRK1, exhibited strong inward rectification and was blocked by divalent cations. However, hIRK differed from IRK1 at the single-channel level: hIRK had a single-channel conductance of 36 pS compared with 21 pS for IRK1. We have identified single channels of 41, 35, 21, and 9 pS in recordings from dispersed human atrial myocytes. However, none of these atrial inward rectifiers exhibited single-channel properties exactly like those of cloned hIRK expressed in oocytes. Our findings suggest that the cardiac IK1 in human atrial myocytes is composed of multiple inwardly rectifying channels distinguishable on the basis of single-channel conductance, each of which may be the product of a different gene.

Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.

Inwardly rectifying K+ channels (IRKs) conduct current preferentially in the inward direction. This inward rectification has two components: voltage-dependent blockade by intracellular Mg2+ (Mg2+i) and intrinsic gating. Two members of this channel family, IRK1 (ref. 10) and ROMK1 (ref. 11), differ markedly in affinity for Mg2+i (ref. 12). We found that IRK1 and ROMK1 differ in voltage-dependent gating and searched for the gating structure by large-scale and site-directed mutagenesis. We found that a single amino-acid change within the putative transmembrane domain M2, aspartate (D) in IRK1 to the corresponding asparagine (N) in ROMK1, controls the gating phenotype. Mutation D172N in IRK1 produced ROMK1-like gating whereas the reverse mutation in ROMK1--N171D--produced IRK1-like gating. Thus, a single negatively charged residue seems to be a crucial determinant of gating.

Inwardly rectifying potassium (K+) channels (IRKs) maintain the resting membrane potential of cells and permit prolonged depolarization, such as during the cardiac action potential. Inward rectification may result from block of the ion conduction pore by intracellular magnesium (Mgi2+). Two members of this family, IRK1 and ROMK1, which share 40 percent amino acid identity, differ markedly in single-channel K+ conductance and sensitivity to block by Mgi2+. The conserved H5 regions were hypothesized to determine these pore properties because they have this function in voltage-dependent K+ channels and in cyclic nucleotide-gated channels. However, exchange of the H5 region between IRK1 and ROMK1 had no effect on rectification and little or no effect on K+ conductance. By contrast, exchange of the amino- and carboxyl-terminal regions together transferred Mg2+ blockade and K+ conductance of IRK1 to ROMK1. Exchange of the carboxyl but not the amino terminus had a similar effect. Therefore, the carboxyl terminus appears to have a major role in specifying the pore properties of IRKs.