Mutations of the S4‐S5 linker alter activation properties of HERG potassium channels expressed in <i>Xenopus</i> oocytes

Sanguinetti, Michael C.; Xu, Qingyang

doi:10.1111/j.1469-7793.1999.667ad.x

Cited by 147 publications

(176 citation statements)

References 26 publications

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“…Deactivation of the HERG channels is strongly dependent on the initial N-terminal residues that constitute the PAS domain (39,40). Previous studies from several laboratories have confirmed that the interaction of the PAS domain with the channel core, including the S4 region (41) and/or the S4-S5 linker (42), determines the slow deactivation kinetics of the HERG channel. The PAS domain is identical in erg1-sm and HERG and suggests that the faster deactivation process may be related to the interaction with the S4 domain.…”

Section: Discussionmentioning

confidence: 99%

Cloning and Functional Characterization of the Smooth Muscle Ether-a-go-go-related Gene K+ Channel

Shoeb¹,

Malykhina²,

Akbarali

2003

Journal of Biological Chemistry

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The human ether-a-go-go-related gene (HERG) product forms the pore-forming subunit of the delayed rectifier K ؉ channel in the heart. Unlike the cardiac isoform, the erg K ؉ channels in native smooth muscle demonstrate gating properties consistent with a role in maintaining resting potential. We have cloned the smooth muscle isoform of HERG, denoted as erg1-sm, from human and rabbit colon. erg1-sm is truncated by 101 amino acids in the C terminus due to a single nucleotide deletion in the 14th exon. Sequence alignment against HERG showed a substitution of alanine for valine in the S4 domain. When expressed in Xenopus oocytes, erg1-sm currents had much faster activation and deactivation kinetics compared with HERG.Step depolarization positive to ؊20 mV consistently produced a transient outward component. The threshold for activation of erg1-sm was ؊60 mV and steady-state conductance was ϳ10-fold greater than HERG near the resting potential of smooth muscle. Site-directed mutagenesis of alanine to valine in the S4 region of erg1-sm converted many of the properties to that of the cardiac HERG, including shifts in the voltage dependence of activation and slowing of deactivation. These studies define the functional role of a novel isoform of the ethera-go-go-related gene K ؉ channel in smooth muscle.The human ether-a-go-go related gene (HERG) 1 encodes for a K ϩ channel that is essential for normal repolarization of the cardiac action potential. HERG was originally cloned from the human hippocampal cDNA library by homology to the Drosophila K ϩ channel gene, eag (1), and according to the latest International Union of Pharmacology (IUPHAR) nomenclature has been termed as Kv 11.1. It is strongly expressed in the mammalian heart, and inherited mutations in this gene cause one form of long Q-T syndrome, LQT2 (2-4). HERG forms the pore-forming subunit of the rapidly activating delayed rectifier K ϩ current, IK r , in native cardiac myocytes (5, 6), and heterologous expression of the cardiac HERG channel in Xenopus oocytes and mammalian cells (7,8) has demonstrated the inwardly rectifying properties of this current. HERG channels slowly activate on depolarization and demonstrate fast inactivation at positive potentials, resulting in small outward currents at potentials positive to 0 mV; hence, inward rectification (9, 10). On repolarization close to resting potentials, large outward tail currents occur that slowly deactivate. These features of HERG channel dictate its role in repolarization of the cardiac action potential and frequency-dependent modulation of the action potential duration (11). Recent studies also suggest that an inwardly rectifying K ϩ conductance that is active near the resting membrane potential of gastrointestinal smooth muscle cells (12, 13), pituitary cells (14 -16), carotid body (15-17), and microglia (18) has properties similar to that of HERG channels. In these cells HERG conductance has been directly demonstrated in single cells, and in esophageal and stomach smooth muscle the presence of a "wind...

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Section: Discussionmentioning

confidence: 99%

Cloning and Functional Characterization of the Smooth Muscle Ether-a-go-go-related Gene K+ Channel

Shoeb¹,

Malykhina²,

Akbarali

2003

Journal of Biological Chemistry

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“…Mutations were introduced into the HERG K ϩ channel (13) by using sitedirected mutagenesis as described previously (14). Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe (Boehringer Mannheim) after linearization of the expression construct with EcoRI.…”

Section: Methodsmentioning

confidence: 99%

“…Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe (Boehringer Mannheim) after linearization of the expression construct with EcoRI. Isolation and maintenance of Xenopus oocytes and cRNA injection were performed as described previously (14)(15)(16).…”

Section: Methodsmentioning

confidence: 99%

A structural basis for drug-induced long QT syndrome

Mitcheson

Chen

Lin

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

860

540

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L ong QT syndrome (LQT) is an abnormality of cardiac muscle repolarization that predisposes affected individuals to a ventricular arrhythmia that can degenerate into ventricular fibrillation and cause sudden death (1). The cellular mechanism of the lengthened QT interval recorded on the body surface electrocardiogram is prolonged ventricular action potentials. Recent genetic discoveries have determined that the molecular mechanism of inherited LQT is mutations in one of several genes [e.g., HERG (2), KCNE2 (3)] that encode ion channel subunits important for normal repolarization of the ventricle. HERG encodes the pore-forming subunits of channels that conduct the rapid delayed rectifier K ϩ current (I Kr ; refs. 4 and 5). LQT also can be acquired as a side effect of treatment with commonly used medications, including some antiarrhythmic, antihistamine, antibiotic, psychoactive, and gastrointestinal prokinetic agents (6, 7). Although drug-induced LQT theoretically could result from reduction of any voltage-gated K ϩ current that contributes to ventricular repolarization, all known drugs with this side effect preferentially block I Kr (1,8,9). It is unclear why so many structurally diverse compounds block HERG channels, but this undesirable side effect now is recognized as a major hurdle in the development of new and safe drugs (9, 10). Previous studies have characterized single residues of HERG that when mutated altered the sensitivity of the channel to block by the methanesulfonanilide antiarrhythmic drug dofetilide. However, these residues are believed to alter drug binding by an allosteric effect related to the loss of inactivation gating (11). A more recent study found that mutation of a Phe located in the S6 domain of HERG decreased block by dofetilide and quinidine (12). However, it is unlikely that a single residue could confer drug sensitivity. Thus, despite the obvious clinical importance of HERG channel block to acquired LQT, the structural basis of the high-affinity drug binding site has not been adequately characterized. Materials and Methods Mutagenesis and Channel Expression in Oocytes.Mutations were introduced into the HERG K ϩ channel (13) by using sitedirected mutagenesis as described previously (14). Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe (Boehringer Mannheim) after linearization of the expression construct with EcoRI. Isolation and maintenance of Xenopus oocytes and cRNA injection were performed as described previously (14-16).Voltage Clamp. The two-microelectrode voltage clamp technique (17) was used to record membrane currents in oocytes 2-4 days after cRNA injection as previously described (18). To attenuate endogenous chloride currents, Cl Ϫ was replaced with Mes (2-[N-morpholino]ethanesulfonic acid) in the external solution that contained 96 mM NaMes͞2 mM KMes͞2 mM CaMes 2 ͞5 mM Hepes͞1 mM MgCl 2 adjusted to pH 7.6 with methane sulfonic acid. In some experiments, KMes was increased to 96 mM with a similar reduction in NaMes.MK-499 was supplied by Me...

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“…Voltage Clamp of Oocytes-Isolation and maintenance of Xenopus laevis oocytes and cRNA injections were performed as described (16).…”

Section: Methodsmentioning

confidence: 99%

Physicochemical Features of the hERG Channel Drug Binding Site

Ghanta

Kauffman

et al. 2004

Journal of Biological Chemistry

262

238

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Blockade of hERG K؉ channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the twodimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltagegated K ؉ channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site. Long QT syndrome (LQTS)1 is a disorder of ventricular repolarization that predisposes affected individuals to cardiac arrhythmia and sudden death. Inherited LQTS is caused by mutations in K ϩ or Na ϩ ion channel genes or ankyrin-B (1, 2). Acquired LQTS is more common and can be induced as an unintended and rare side effect of treatment with many structurally diverse medications. In the past few years, several commonly used drugs (e.g. terfenadine, cisapride, sertindole, thioridazine, grepafloxacin) were withdrawn from the market, or their approved use was severely restricted, when it was discovered that they caused arrhythmia or were associated with unexplained sudden death, albeit very infrequently (3). The molecular basis of drug-induced LQTS is block of human ether-a-go-go related gene (hERG) channels that conduct I Kr , the rapid delayed rectifier K ϩ current important for repolarization of cardiac action potentials (4, 5). A reduction in I Kr prolongs the action potential duration of ventricular myocytes, lengthens the QT interval and increases dispersion as measured by ECG recordings, and increases the risk of torsades de pointes, a ventricular tachyarrhythmia that can degenerate into fibrillation and cause sudden death. In a laboratory setting, it is possible to induce arrhythmia in animals with drugs that block voltage-gated K ϩ (Kv) channels other than hERG. However, in clinical practice, drug-induced LQTS is always attributable...

show abstract

Mutations of the S4‐S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes

Cited by 147 publications

References 26 publications

Cloning and Functional Characterization of the Smooth Muscle Ether-a-go-go-related Gene K+ Channel

Cloning and Functional Characterization of the Smooth Muscle Ether-a-go-go-related Gene K+ Channel

A structural basis for drug-induced long QT syndrome

Physicochemical Features of the hERG Channel Drug Binding Site

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