Individual subunits contribute independently to slow gating of bovine EAG potassium channels.
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The bovine ether a go-go gene encodes a delayed rectifier potassium channel. In contrast to other delayed rectifiers, its activation kinetics is largely determined by the holding potential and the concentration of extracellular Mg2+, giving rise to slowly activating currents with a characteristic sigmoidal rising phase. Replacement of a single amino acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild type, the half-activation voltage of this mutant was shifted by more than 30 mV to more negative potentials. We used dimeric and tetrameric constructs of the bovine eag1 gene to analyze channels with defined stoichiometry of mutated and wild-type subunits within the tetrameric channel complexes. With increasing numbers of mutated subunits, the channel activation was progressively accelerated, and the sigmoidicity of the current traces was reduced. Based on a quantitative analysis, we show that the slow gating, typical for EAG channels, is mediated by independent conformational transitions of individual subunits, which gain their voltage dependence from the S4 segment. At a given voltage, external Mg2+ increases the probability of a channel subunit to be in the slowly activating conformation, whereas mutation L322H strongly reduces this probability. (+info)
Ion channels: structure of a molecular brake.
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A combination of crystallographic and mutagenesis studies on the HERG K+ channel, a key determinant of cardiac excitability, has suggested how the protein's extramembraneous amino-terminal domain might act as a 'molecular brake' that slows down channel deactivation. (+info)
Overexpression of a human potassium channel suppresses cardiac hyperexcitability in rabbit ventricular myocytes.
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The high incidence of sudden death in heart failure may reflect abnormalities of repolarization and heightened susceptibility to arrhythmogenic early afterdepolarizations (EADs). We hypothesized that overexpression of the human K+ channel HERG (human ether-a-go-go-related gene) could enhance repolarization and suppress EADs. Adult rabbit ventricular myocytes were maintained in primary culture, which suffices to prolong action potentials and predisposes to EADs. To achieve efficient gene transfer, we created AdHERG, a recombinant adenovirus containing the HERG gene driven by a Rous sarcoma virus (RSV) promoter. The virally expressed HERG current exhibited pharmacologic and kinetic properties like those of native IKr. Transient outward currents in AdHERG-infected myocytes were similar in magnitude to those in control cells, while stimulated action potentials (0.2 Hz, 37 degrees C) were abbreviated compared with controls. The occurrence of EADs during a train of action potentials was reduced by more than fourfold, and the relative refractory period was increased in AdHERG-infected myocytes compared with control cells. Gene transfer of delayed rectifier potassium channels represents a novel and effective strategy to suppress arrhythmias caused by unstable repolarization. (+info)
C-terminal HERG mutations: the role of hypokalemia and a KCNQ1-associated mutation in cardiac event occurrence.
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BACKGROUND: The long-QT syndrome (LQTS) is a genetically heterogeneous disease in which 4 genes encoding ion-channel subunits have been identified. Most of the mutations have been determined in the transmembrane domains of the cardiac potassium channel genes KCNQ1 and HERG. In this study, we investigated the 3' part of HERG for mutations. METHODS AND RESULTS: New specific primers allowed the amplification of the 3' part of HERG, the identification of 2 missense mutations, S818L and V822 M, in the putative cyclic nucleotide binding domain, and a 1-bp insertion, 3108+1G. Hypokalemia was a triggering factor for torsade de pointes in 2 of the probands of these families. Lastly, in a large family, a maternally inherited G to A transition was found in the splicing donor consensus site of HERG, 2592+1G-A, and a paternally inherited mutation, A341E, was identified in KCNQ1. The 2 more severely affected sisters bore both mutations. CONCLUSIONS: The discovery of mutations in the C-terminal part of HERG emphasizes that this region plays a significant role in cardiac repolarization. Clinical data suggests that these mutations may be less malignant than mutations occurring in the pore region, but they can become clinically significant in cases of hypokalemia. The first description of 2 patients with double heterozygosity associated with a dramatic malignant phenotype implies that genetic analysis of severely affected young patients should include an investigation for >1 mutation in the LQT genes. (+info)
Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations.
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We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance. (+info)
Human ether-a-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors.
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Human ether-a-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-a-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery. (+info)
Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of HERG potassium channels accelerate channel deactivation.
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Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome, an inherited disorder of cardiac repolarization that predisposes affected individuals to life-threatening arrhythmias. HERG encodes the cardiac rapid delayed rectifier potassium channel that mediates repolarization of ventricular action potentials. In this study, we used the oocyte expression system and voltage clamp techniques to determine the functional consequences of eight long QT syndrome-associated mutations located in the amino-terminal region of HERG (F29L, N33T, G53R, R56Q, C66G, H70R, A78P, and L86R). Mutant subunits formed functional channels with altered gating properties when expressed alone in oocytes. Deactivation was accelerated by all mutations. Some mutants shifted the voltage dependence of channel availability to more positive potentials. Voltage ramps indicated that fast deactivation of mutant channels would reduce outward current during the repolarization phase of the cardiac action potential and cause prolongation of the corrected QT interval, QTc. The amino-terminal region of HERG was recently crystallized and shown to possess a Per-Arnt-Sim (PAS) domain. The location of these mutations suggests they may disrupt the PAS domain and interfere with its interaction with the S4-S5 linker of the HERG channel. (+info)
Functional analysis of a mouse brain Elk-type K+ channel.
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Members of the Ether a go-go (Eag) K+ channel subfamilies Eag, Erg, and Elk are widely expressed in the nervous system, but their neural functions in vivo remain largely unknown. The biophysical properties of channels from the Eag and Erg subfamilies have been described, and based on their characteristic features and expression patterns, Erg channels have been associated with native currents in the heart. Little is known about the properties of channels from the Elk subfamily. We have identified a mouse gene, Melk2, that encodes a predicted polypeptide with 48% amino acid identity to Drosophila Elk but only 40 and 36% identity with mouse Erg (Merg) and Eag (Meag), respectively. Melk2 RNA appears to be expressed at high levels only in brain tissue. Functional expression of Melk2 in Xenopus oocytes reveals large, transient peaks of current at the onset of depolarization. Like Meag currents, Melk2 currents activate relatively quickly, but they lack the nonsuperimposable Cole-Moore shift characteristic of the Eag subfamily. Melk2 currents are insensitive to E-4031, a class III antiarrhythmic compound that blocks the Human Ether-a-go-go-Related Gene (HERG) channel and its counterpart in native tissues, IKr. Melk2 channels exhibit inward rectification because of a fast C-type inactivation mechanism, but the slower rate of inactivation and the faster rate of activation results in less inward rectification than that observed in HERG channels. This characterization of Melk currents should aid in identification of native counterparts to the Elk subfamily of channels in the nervous system. (+info)