High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.     

Inhibition of HERG potassium channel current by the class 1a antiarrhythmic agent disopyramide

The Class 1a antiarrhythmic drug disopyramide (DISO) is associated with 'acquired' prolongation of the QT interval of the electrocardiogram (ECG). This potentially proarrhythmic effect is likely to reflect drug actions on ion channels involved in ventricular action potential repolarisation. In this study, we examined the effects of DISO on potassium channels encoded by HERG, as this K channel type has been implicated in both congenital and acquired long-QT syndromes (LQTS). Chinese hamster ovary cells were transiently transfected with HERG cDNA for subsequent whole cell patch clamp recording. HERG tail currents recorded at -40 mV following test pulses to +30 mV were inhibited in a dose-dependent fashion by DISO concentrations within the clinical range (IC50 = 7.23 +/- 0.72 microM; mean +/- SEM). Experiments with 10 microM DISO indicated that the degree of HERG blockade showed some voltage dependence. Further data obtained using an 'envelope of tails' protocol (pulse potential +40 mV) were consistent with a significant role for open-channel blockade at lower drug concentrations. At higher concentrations it is possible that blockade may have involved drug binding to both resting and open channels. Inhibition of the inactivation-deficient mutant HERG-S631A was comparable to that seen for wild-type HERG. Therefore, channel inactivation was not obligatory for DISO to exert its effect. Native delayed rectifier tail currents from rabbit isolated ventricular myocytes were also inhibited by DISO. We conclude (a) that DISO inhibits HERG encoded potassium channels at clinically relevant concentrations and (b) that this action may constitute the molecular basis for acquired LQTS associated with this drug.     

Fast inactivation causes rectification of the IKr channel

The mechanism of rectification of HERG, the human cardiac delayed rectifier K+ channel, was studied after heterologous expression in Xenopus oocytes. Currents were measured using two-microelectrode and macropatch voltage clamp techniques. The fully activated current- voltage (I-V) relationship for HERG inwardly rectified. Rectification was not altered by exposing the cytoplasmic side of a macropatch to a divalent-free solution, indicating this property was not caused by voltage-dependent block of outward current by Mg2+ or other soluble cytosolic molecules. The instantaneous I-V relationship for HERG was linear after removal of fast inactivation by a brief hyperpolarization. The time constants for the onset of and recovery from inactivation were a bell-shaped function of membrane potential. The time constants of inactivation varied from 1.8 ms at +50 mV to 16 ms at -20 mV; recovery from inactivation varied from 4.7 ms at -120 mV to 15 ms at -50 mV. Truncation of the NH2-terminal region of HERG shifted the voltage dependence of activation and inactivation by +20 to +30 mV. In addition, the rate of deactivation of the truncated channel was much faster than wild-type HERG. The mechanism of HERG rectification is voltage-gated fast inactivation. Inactivation of channels proceeds at a much faster rate than activation, such that no outward current is observed upon depolarization to very high membrane potentials. Fast inactivation of HERG and the resulting rectification are partly responsible for the prolonged plateau phase typical of ventricular action potentials.     

酮色林对人类ether-a-go-go相关基因钾通道的阻断作用

采用双电极电压钳技术,研究酮色林对表达在非洲爪蟾卵母细胞上的野生型和Y652突变型人类ether-a-go-go相关基因(human ether-a-go-go-related gene,HERG)钾通道的阻断效应,观测HERG通道的分子位点特性改变对其阻断效应的影响.结果显示,酮色林以电压依赖性和浓度依赖性的方式阻断野生型的HERG钾通道电流.尾电流包裹程序记录电流显示酮色林对HERG钾通道微小的张力性阻断.阻断特征符合对开放状态通道的阻断特征.酮色林也能调节失活状态的HERG钾通道.位于孔道S6区的氨基酸位点突变Y652A和Y652R可显著减弱酮色林对HERG通道的阻断作用.同野生犁HERG钾通道的阻断相比,Y652A突变使阻断的IC50提高72倍,而Y652R突变使阻断的IC50提高53倍.Y652A和Y652R的阴断效应之间没有明显的差别.以上结果提示,酮色林优先阻断开放状态的HERG钾通道,而Y652是酮色林与通道结合的关键位点之一.     

The binding site for channel blockers that rescue misprocessed human long QT syndrome type 2 ether-a-gogo-related gene (HERG) mutations.

Mutations in the human ether-a-gogo-related gene (HERG) K(+) channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduce I(Kr) currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K(+) channels without block.     

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

Effect of sibutramine HCl on cardiac hERG K+ channel

Common clinically used drugs block the delayed rectifier K(+) channels and prolong the cardiac action potential duration associated with long QT syndrome. Here, we investigated the mechanism of hERG K(+) channel current (I(hERG)) blockade expressed in HEK-293 cells by sibutramine HCl, a serotonin-norepinephrine reuptake inhibitor. Sibutramine HCl inhibited I (hERG) in a concentration-dependent manner with the half-maximal inhibitory concentration (IC(50)) value of 2.5 microM at -40 mV. I(hERG) inhibition by sibutramine HCl showed weak voltage dependency, but the time-dependence of I(hERG) inhibition was developed relatively rapidly on membrane depolarization. On hERG channel gating for the S6 and pore regions, the S6 residue hERG mutant Y652A and F656A largely reduced the blocking potency of I(hERG), unlike the pore-region mutants T623A and S624A. These results indicate that sibutramine HCl preferentially inhibits the hERG potassium channel through the residue Y652 and F656, in a supratherapeutic concentration should be avoided by patients with high susceptibility for cardiac arrhythmia.     

Enhancement of HERG K(+) currents by Cd(2+) destabilization of the inactivated state

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 &mgr;M) of extracellular Cd(2+) increased outward currents through HERG channels; 200 &mgr;M Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 &mgr;M did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >/=500 &mgr;M shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.     

Molecular determinants of voltage-dependent human ether-a-go-go related gene (HERG) K+ channel block

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K(+) channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC(50) was 8.4 +/- 0.9 microm when assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC(50) > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-pi and pi-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.     

Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate

Deactivation of voltage-gated potassium (K(+)) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC(50) = 32 nM) of HERG K(+) channels. This bulky compound (7 x 20 A) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 microM MK-499 to occur with time constants of 10.5 and 52.7 s at -160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K(+) channel.     

Preferential closed channel blockade of HERG potassium currents by chemically synthesised BeKm-1 scorpion toxin

The scorpion toxin peptide BeKm-1 was synthesised by fluorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's effects on heterologous human ether-a-go-go-related gene potassium current (I(HERG)) in HEK293 cells were assessed using 'whole-cell' patch clamp. Blockade of I(HERG) by BeKm-1 was concentration-dependent, temperature-dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use- and frequency-dependence. Blockade by BeKm-1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm-1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs.     

Inactivation of BK channels mediated by the NH(2) terminus of the beta3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade

A family of auxiliary beta subunits coassemble with Slo alpha subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The beta subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The beta3b auxiliary subunit, when coexpressed with the Slo alpha subunit, results in a particularly rapid ( approximately 1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the beta3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism (C(n) <---> O(n) <---> O(*)(n) <---> I(n)) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.     

Effects of carvedilol on transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes

Carvedilol is a beta- and alpha(1)-adrenoceptor antagonist. It is widely used in the treatment of cardiovascular diseases including atrial arrhythmias. However, it is unclear whether carvedilol may affect the repolarization currents, transient outward K(+) current (I(to)) and ultra-rapid delayed rectifier K(+) current (I(Kur)) in the human atrium. The present study evaluated effects of carvedilol on I(to) and I(Kur) in isolated human atrial myocytes by whole-cell patch-clamp recording technique. We found that carvedilol reversibly inhibited I(to) and I(Kur) in a concentration-dependent manner. Carvedilol (0.3 microM) suppressed I(to) from 9.2+/-0.5 pA/pF to 4.8+/-0.5 pA/pF (P<0.01) and I(Kur) from 3.6+/-0.5 pA/pF to 1.9+/-0.3 pA/pF (P<0.01) at +50 mV. I(to) was inhibited in a voltage-dependent manner, being significantly attenuated at test potentials from +10 to +50 mV, whereas the inhibition of I(Kur) was independent. The concentration giving a 50% inhibition was 0.50 microM for I(to) and 0.39 microM for I(Kur). Voltage-dependence of activation, inactivation and time-dependent recovery from inactivation of I(to) were not altered by carvedilol. However, time to peak and time-dependent inactivation of I(to) were significantly accelerated, indicating an open channel blocking action. The findings indicate that carvedilol significantly inhibits the major repolarization K(+) currents I(to) and I(Kur) in human atrial myocytes.     

Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels

K(+) channels encoded by the human ether-à-go-go-related gene (HERG) are distinguished from most other voltage-gated K(+) channels by an unusually slow deactivation process that enables cardiac I(Kr), the corresponding current in ventricular cells, to contribute to the repolarization of the action potential. When the first 16 amino acids are deleted from the amino terminus of HERG, the deactivation rate is much faster (Wang, J., M.C. Trudeau, A.M. Zappia, and G.A. Robertson. 1998. J. Gen. Physiol. 112:637-647). In this study, we determined whether the first 16 amino acids comprise a functional domain capable of slowing deactivation. We also tested whether this "deactivation subdomain" slows deactivation directly by affecting channel open times or indirectly by a blocking mechanism. Using inside-out macropatches excised from Xenopus oocytes, we found that a peptide corresponding to the first 16 amino acids of HERG is sufficient to reconstitute slow deactivation to channels lacking the amino terminus. The peptide acts as a soluble domain in a rapid and readily reversible manner, reflecting a more dynamic regulation of deactivation than the slow modification observed in a previous study with a larger amino-terminal peptide fragment (Morais Cabral, J.H., A. Lee, S.L. Cohen, B.T. Chait, M. Li, and R. Mackinnon. 1998. Cell. 95:649-655). The slowing of deactivation by the peptide occurs in a dose-dependent manner, with a Hill coefficient that implies the cooperative action of at least three peptides per channel. Unlike internal TEA, which slows deactivation indirectly by blocking the channels, the peptide does not reduce current amplitude. Nor does the amino terminus interfere with the blocking effect of TEA, indicating that the amino terminus binding site is spatially distinct from the TEA binding site. Analysis of the single channel activity in cell-attached patches shows that the amino terminus significantly increases channel mean open time with no alteration of the mean closed time or the addition of nonconducting states expected from a pore block mechanism.We propose that the four amino-terminal deactivation subdomains of the tetrameric channel interact with binding sites uncovered by channel opening to specifically stabilize the open state and thus slow channel closing.     

Block of wild-type and inactivation-deficient cardiac sodium channels IFM/QQQ stably expressed in mammalian cells

The role of inactivation as a central mechanism in blockade of the cardiac Na(+) channel by antiarrhythmic drugs remains uncertain. We have used whole-cell and single channel recordings to examine the block of wild-type and inactivation-deficient mutant cardiac Na(+) channels, IFM/QQQ, stably expressed in HEK-293 cells. We studied the open-channel blockers disopyramide and flecainide, and the lidocaine derivative RAD-243. All three drugs blocked the wild-type Na(+) channel in a use-dependent manner. There was no use-dependent block of IFM/QQQ mutant channels with trains of 20 40-ms pulses at 150-ms interpulse intervals during disopyramide exposure. Flecainide and RAD-243 retained their use-dependent blocking action and accelerated macroscopic current relaxation. All three drugs reduced the mean open time of single channels and increased the probability of their failure to open. From the abbreviation of the mean open times, we estimated association rates of approximately 10(6)/M/s for the three drugs. Reducing the burst duration contributed to the acceleration of macroscopic current relaxation during exposure to flecainide and RAD-243. The qualitative differences in use-dependent block appear to be the result of differences in drug dissociation rate. The inactivation gate may play a trapping role during exposure to some sodium channel blocking drugs.     

K(+) currents responsible for repolarization in mouse ventricle and their modulation by FK-506 and rapamycin

Modulation of mouse ventricular action potentials and K(+) currents was examined using the whole cell patch-clamp technique. The composite mouse ventricular K(+) current (consisted of an outward transient followed by a slowly decaying sustained component. Use of the K(+) channel blockers tetraethylammonium and 4-aminopyridine and a transgenic mouse model revealed three pharmacologically and kinetically distinct currents: I(to), which contributed to the transient component; I(K), which contributed to the sustained component; and a slowly activating current (I(slow)), which contributed to both components. The immunosuppressant FK-506 increased action potential duration at 90% repolarization by 66.7% by decreasing the sustained component (-48% at +60 mV) and prolonging recovery from inactivation (by 26% at 200 ms) of the transient component. These effects were isolated to I(K) and I(to), respectively. Rapamycin had strikingly similar effects on these currents. Both FK-506 and rapamycin are known to target the immunophilin FKBP12. Thus we conclude that FKBP12 modulates specific mouse K(+) channels, and thus the mouse ventricular action potential, by interacting directly with K(+) channel proteins or with other associated regulatory proteins.     

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

Voltage-dependent K(+) channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel's selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K(+) channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca(2+) or Ba(2+), suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K(+)] (47 mV per 10-fold increase in [K(+)]), suggesting that K(+) binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K(+) ≈ Rb(+) > Cs(+) > Na(+) > Li(+) ≈ NMG(+). Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K(+)] using kinetic schemes in which the open-conductive state is stabilized by K(+) binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K(+) dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K(+)-sensitive inactivation gating, a property that may be common to other K(+) channels.     

Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.