Figure - available via license: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International
Content may be subject to copyright.
Baseline steady-state inactivation of WT, S624A, and S624T HERG channels. A “triple-pulse” voltage clamp protocol (Smith et al., 1996; Spector et al., 1996) was used to assess the voltage dependence of channel availability in 0 Na+o, 0 K+o. A–C show typical families of currents at the test potential and a subsequent step to 30 mV for WT HERG (closed squares), S624A (open circles), and S624T (dotted triangles), respectively. Peak current observed immediately after stepping to 30 mV was normalized to the largest peak current recorded in the same cell. Data from several cells (n = 4 for each channel) are summarized in D. At test potentials greater than −20 mV for S624A and greater than −10 mV for S624T, normalized current was significantly greater than that observed for WT HERG (P < 0.05), consistent with impaired inactivation relative to WT.
Source publication
Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG curren...
Citations
... To determine whether the nature of trafficking plays a role in RDV-mediated mRNA increases, we compared the effects of RDV treatment (10 µM, 24 h) on mRNA levels between trafficking competent mutant S624T and trafficking defective N629D hERG channels. S624T is a mutant hERG channel that traffics normally and displays altered sensitivity to extracellular Na + and K + levels (Guo et al., 2009;Massaeli et al., 2010;Mullins et al., 2002;Mullins et al., 2004). N629D hERG is a human missense LQT2 mutation (Satler et al., 1998). ...
The human ether-a-go-go-related gene (hERG) encodes for the pore-forming subunit of the channel that conducts the rapidly activating delayed K+ current (IKr) in the heart. The hERG channel is important for cardiac repolarization, and reduction of its expression in the plasma membrane due to mutations causes long-QT syndrome type 2 (LQT2). As such, promoting hERG membrane expression is a strategy to rescue mutant channel function. In the present study, we applied patch clamp, Western blots, immunocytochemistry, and Quantitative Reverse Transcription PCR (RT-qPCR) techniques to investigate the rescue effects of two drugs, remdesivir and lumacaftor, on trafficking defective mutant hERG channels. As our group has recently reported that the antiviral drug remdesivir increases wild-type (WT) hERG current and surface expression, we studied the effects of remdesivir on trafficking defective LQT2-causing hERG mutants G601S and R582C expressed in HEK293 cells. We also investigated the effects of lumacaftor, a drug used to treat cystic fibrosis that promotes CFTR protein trafficking and has been shown to rescue membrane expression of some hERG mutations. Our results show that neither remdesivir nor lumacaftor rescued the current or cell-surface expression of homomeric mutants G601S and R582C. However, remdesivir decreased while lumacaftor increased the current and cell-surface expression of heteromeric channels formed by WT hERG and mutant G601S or R582C hERG. We concluded that drugs can differentially affect homomeric WT and heteromeric WT+G601S (or WT+R582C) hERG channels. These findings extend our understanding of drug-channel interaction, and may have clinical implications for patients with hERG mutations. Significance Statement Various naturally occurring mutations in a cardiac potassium channel called hERG can impair channel function by decreasing cell-surface channel expression, resulting in cardiac electrical disturbances and even sudden cardiac death. Promotion of cell-surface expression of mutant hERG channels represents a strategy to rescue channel function. This work demonstrates that drugs such as remdesivir and lumacaftor can differently affect homomeric and heteromeric mutant hERG channels, which have biological and clinical implications.
... Step I-V curves for WT, S624A, and Y652A showed maximum step currents at 0 mV, -10 mV, and +10 mV, respectively (Fig 3A, bottom). At +60 mV, S624A had more than twice the relative amount of current compared to WT channels, which indicated possible incomplete inactivation; this mutant channel was previously characterized to have a slower inactivation [53,54]. Boltzmann fits to the activation curves indicated no significant difference in half-maximal activation between S624A, Y652A, and WT ( Fig 3B). ...
... Third, drug unbinding could be reduced in S624A, since this residue has been previously implicated in the dissociation of hERG blockers (e.g., recovery from propafenone-mediated inhibition of hERG was significantly reduced in the S624A mutant [85]). Finally, altered inactivation in the S624A mutant [53,54], may have enhanced inhibition. But this is unlikely to occur since a non-inactivating S620T mutant does not exhibit stronger inhibition [23], and considering that inactivation is removed from tail currents. ...
... Another aspect of R-roscovitine's inhibition of S624A is that there appeared to be some reduction in R-oscovitine block at higher voltages, which was visible in both step and tail IV curves (Fig 4). This, along with the dependence of block on the direction of current (see S1C Fig) are consistent with pore block, but future studies are warranted to explore the mechanisms by which R-roscovitine binds to the S624A pore to modulate the already aberrant S624A gating (see Figs 3 and 4; [53,54]). ...
Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 μM R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine's relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.
... However, a potential contribution of altered hERG channel conductance to the overall effect (as suggested by Wang et al. 1997a) cannot be ruled out, given that single hERG channel conductance is known to vary with [K + ] e (2 pS at 5 mmol/L and 10 pS at 100 mmol/L in Kiehn et al. [1996]). Although I hERG is known to be sensitive to [Na + ] e (Namaguchi et al. 2000;Mullins et al. 2002) (Lu et al. 2001(Lu et al. , 2003McPate et al. 2009;Du et al. 2010). Application of premature stimuli between 100 msec before APD 90 of the initial AP and 190 msec after APD 90 was sufficient to reveal the normal biphasic relationship of I hERG transient amplitude with time late in repolarization/early in diastole (Lu et al. 2001(Lu et al. , 2003McPate et al. 2009;Du et al. 2010). ...
... and changes to [K + ] e in the present study were compensated by concomitant alterations to [Na + ] e , the modulatory effects of [Na + ] e on I hERG amplitude are most marked for [Na + ] e concentrations substantially below 100 mmol/L(Namaguchi et al. 2000;Mullins et al. 2002) and so are unlikely to contribute significantly to observed effects of altering [K + ] e in our experiments.The profile of WT I hERG seen here in normal (4 mmol/L) [K + ] e both during imposed AP clamp commands and in response to premature AP stimuli(Figs 4 and 5)is comparable to that found in prior studies that have used similar paired AP clamp protocols, with maximal I hERG transient amplitude occurring when premature stimuli were applied shortly after the point of 90% complete repolarization of the first AP (APD 90 ) ...
Potassium channels encoded by human ether-à-go-go-related gene (hERG) mediate the cardiac rapid delayed rectifier K+ current (IKr), which participates in ventricular repolarization and has a protective role against unwanted premature stimuli late in repolarization and early in diastole. Ionic current carried by hERG channels (IhERG) is known to exhibit a paradoxical dependence on external potassium concentration ([K+]e), but effects of acute [K+]e changes on the response of IhERG to premature stimulation have not been characterized. Whole-cell patch-clamp measurements of hERG current were made at 37°C from hERG channels expressed in HEK293 cells. Under conventional voltage-clamp, both wild-type (WT) and S624A pore-mutant IhERG during depolarization to +20 mV and subsequent repolarization to −40 mV were decreased when superfusate [K+]e was decreased from 4 to 1 mmol/L. When [K+]e was increased from 4 to 10 mmol/L, pulse current was increased and tail IhERG was decreased. Increasing [K+]e produced a +10 mV shift in voltage-dependent inactivation of WT IhERG and slowed inactivation time course, while lowering [K+]e from 4 to 1 mmol/L produced little change in inactivation voltage dependence, but accelerated inactivation time course. Under action potential (AP) voltage-clamp, lowering [K+]e reduced the amplitude of IhERG during the AP and suppressed the maximal IhERG response to premature stimuli. Raising [K+]e increased IhERG early during the AP and augmented the IhERG response to premature stimuli. Our results are suggestive that during hypokalemia not only is the contribution of IKr to ventricular repolarization reduced but its ability to protect against unwanted premature stimuli also becomes impaired.
... While this mechanism of current inhibition has not been described previously for T-channels, similar finding was reported for voltage-gated Na þ and voltage-gated K þ channels. Specifically, Mullins et al. (2002) presented a model where both hastening of recovery from inactivation of hERG K þ channels and accelerated entry into inactivated states are induced by Na þ ions. Similarly, Szendroedi et al. (2007) reported that Cd 2þ ions blocked currents though recombinant voltage-gated Na þ channels by shifting steady-state inactivation to hyperpolarized potentials and concomitantly speeding the recovery from inactivation. ...
... The results described above indicate that the NS1643-induced increases in macroscopic Kv11.3 conductance in the fullyactivated state cannot be explained by reduced inactivation and must involve other mechanisms. For Kv11.1a, the classical HERG channel, external Ca 2+ as well as external Na + have been found to inhibit the channel and external binding sites for these ions have been discussed [26,27]. To test whether NS1643 might exert part of its activating effects by a relief of the Kv11 current inhibition produced by these external cations, experiments were performed in external zero Na + or low Ca 2+ solution. ...
... It has been shown that rises in external K + enhance the conductance of all three Kv11 channels [2]. The mechanism of the unusual K + dependence of Kv11.1 has been explained by a channel blocking effect of external Na + ions which is relieved by rises in external K + [27,39]. We have now found that NS1643 is able to increase fully-activated Kv11.3 currents in the absence of external Na + , suggesting that the drug-induced increase in macroscopic Kv11.3 channel conductance is not mediated by a decrease in the binding affinity of Na + ions. ...
NS1643 is one of the small molecule HERG (Kv11.1) channel activators and has also been found to increase erg2 (Kv11.2) currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3) channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+) or Ca(2+). At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.
... Membrane potential was stepped from −80 mV to a test potential between −70 and 100 mV, in intervals of 10 mV, for 2 s, followed by step to −50 mV. The HERG characteristic rapid rise in the tails of current account for a very fast recovery from inactivation and a slower inactivation(378). ...
Potassium channels are particularly important in determining the shape and duration of the action potential, controlling the membrane potential, modulating hormone secretion, epithelial function and, in the case of those K(+) channels activated by Ca(2+), damping excitatory signals. The multiplicity of roles played by K(+) channels is only possible to their mammoth diversity that includes at present 70 K(+) channels encoding genes in mammals. Today, thanks to the use of cloning, mutagenesis, and the more recent structural studies using x-ray crystallography, we are in a unique position to understand the origins of the enormous diversity of this superfamily of ion channels, the roles they play in different cell types, and the relations that exist between structure and function. With the exception of two-pore K(+) channels that are dimers, voltage-dependent K(+) channels are tetrameric assemblies and share an extremely well conserved pore region, in which the ion-selectivity filter resides. In the present overview, we discuss in the function, localization, and the relations between function and structure of the five different subfamilies of K(+) channels: (a) inward rectifiers, Kir; (b) four transmembrane segments-2 pores, K2P; (c) voltage-gated, Kv; (d) the Slo family; and (e) Ca(2+)-activated SK family, SKCa. © 2012 American Physiological Society. Compr Physiol 2:2087-2149, 2012.
... Changing the external [K ϩ ] relieved proton inhibition with a K d of ϳ1 mM (Fig. 5, A and C) consistent with a proton binding site within the pore. Detailed studies have shown that Na ϩ binds within the outer pore of hERG channels when the external [K ϩ ] is low (37,40). These studies showed that external K ϩ relieved Na ϩ block with a K d ϳ3 mM, resulting in a labile channel conductance within the physiological range of [K ϩ ]. ...
... This is supported by the observation that proton block was relieved by increasing external Na ϩ in the absence of K ϩ (the K d for Na ϩ relief was ϳ56 mM, Fig. 5E). Na ϩ binding within the outer pore of hERG channels was previously shown to hinder the exit of Ba 2ϩ from its binding site deeper within the pore (37). The observation that Na ϩ hinders protons from accessing the pore of the hERG channels supports the conclusion that protons bind within the outer pore. ...
Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.
... Our experiments did not identify which additional mechanism-apart from the leftward shift in activation and slowing of deactivation-is involved in the observed increase in erg1b current amplitude, because erg1b channel inactivation was not affected by NS1643. Other possible mechanisms that could contribute to the drug-induced increase in erg wholecell current are an enhanced maximal channel open probability or a reduced erg channel affinity to external Na ϩ , thus reducing the Na ϩ block suggested to underlie the paradoxical agonist effect of external K ϩ on herg current amplitude (Numaguchi et al., 2000;Mullins et al., 2002). ...
Two different mechanisms leading to increased current have been described for the small-molecule human ether-à-go-go-related gene (herg) activator NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea]. On herg1a channels expressed in Xenopus laevis oocytes, it mainly acts via attenuation of inactivation and for rat (r) erg1b channels expressed in human embryonic kidney (HEK)-293 cells, it strongly shifts the activation curve to the left. We now investigated the NS1643 effects on erg1b channels in more detail and performed comparative experiments with rat and human erg1a in different expression systems. Significant differences were observed between expression systems, but not between the rat and human isoform. In HEK-293 or Chinese hamster ovary (CHO) cells, activation of rat erg1b channels occurred in a dose-dependent manner with a maximum current increase of 300% obtained with 10 μM NS1643. In contrast, the NS1643-induced strong leftward shift in the voltage dependence of activation further increased with higher drug concentration, needed more time to develop, and exhibited use dependence. Coexpression of KCNE1 or KCNE2 did not attenuate this NS1643 effect on erg1 channel activation and did thus not mimic the lower drug potency on this parameter observed in oocytes. NS1643 (10 μM) slowed erg1b channel deactivation and recovery from inactivation without significant changes in activation and inactivation kinetics. With the exception of accelerated activation, NS1643 affected erg1a channels similarly, but the effect was less pronounced than in erg1b or erg1a/1b channels. It is noteworthy that rerg1b and herg1a inactivation estimated from fully activated current voltage relationships were unaltered in the continued presence of 10 μM NS1643 in the mammalian expression systems, indicating qualitative differences from NS1643 effects in X. laevis oocytes.
... A modified ND99 solution (extracellular K þ-free solution) was used in the bath, due to the reported change in selectivity of the same mutant in other Kv channels (27,28). The predictable outward tail currents recorded from WT channels in those conditions (data not shown) were small due to the fact that extracellular sodium potently inhibits hERG channels when extracellular K þ is removed (33,34). Small currents were obtained from the G628S mutant, with inwardly directed tail currents (Fig. 3, A and B), suggesting that they are sodium-dependent. ...
G628S is a mutation in the signature sequence that forms the selectivity filter of the human ether-a-go-go-related gene (hERG) channel (GFG) and is associated with long-QT2 syndrome. G628S channels are known to have a dominant-negative effect on hERG currents, and the mutant is therefore thought to be nonfunctional. This study aims to assess the physiological mechanism that prevents the surface-expressing G628S channels from conducting ions. We used voltage-clamp fluorimetry along with two-microelectrode voltage clamping in Xenopus oocytes to confirm that the channels express well at the surface, and to show that they are actually functional, with activation kinetics comparable to that of wild-type, and that the mutation leads to a reduced selectivity to potassium. Although ionic currents are not detected in physiological solutions, removing extracellular K(+) results in the appearance of an inward Na(+)-dependent current. Using whole-cell patch clamp in mammalian transfected cells, we demonstrate that the G628S channels conduct Na(+), but that this can be blocked by both intracellular and higher-than-physiological extracellular K(+). Using solutions devoid of K(+) allows the appearance of nA-sized Na(+) currents with activation and inactivation gating analogous to wild-type channels. The G628S channels are functionally conducting but are normally blocked by intracellular K(+).
... mV (nϭ5) for S631A. These results are consistent with the previous reports 13, [21][22][23] and indicate that compared to WT channels, the voltage dependence of steady-state inactivation is greatly shifted to the hyperpolarized potentials for the F627Y, slightly shifted to the depolarized potentials for the S624A, and drastically shifted to the depolarized potentials for the S631A. If inactivation is responsible for the reduced I HERG in low K ϩ o , we would expect that mutant channels with a diminished inactivation (depolarized shift of the steadystate inactivation, eg, S631A) would exhibit a reduced sensitivity to 0 mmol/L K ϩ exposure, whereas mutant channels with enhanced inactivation (hyperpolarized shift of the steady-state inactivation, eg, F627Y) would display an increased sensitivity to 0 mmol/L K ϩ exposure. ...
... For example, S624 in HERG corresponds to T75 in KcsA, which constitutes the innermost selectivity filter position of the channel and contributes to the potential ring of hydrogen bonds encircling the inner selectivity filter. 22 F627 in HERG corresponds to Y78 in KcsA, which forms hydrogen bonds with nearby nitrogen atoms of the W. Thus, S624T or F627Y may allow formation of hydrogen bonds in the mutant channel and thus lead to a more rigid channel conformation. We showed that WT/S624T channels displayed K ϩ dependency similar to WT channels ( Figure 6C), suggesting that the mutation in each subunit is required to enhance the channel stability. ...
The human ether-a-go-go-related gene (HERG) encodes the pore-forming subunits of the rapidly activating delayed rectifier potassium channel (I(Kr)) that is important for cardiac repolarization. Dysfunction of HERG causes long QT syndrome (LQTS) which can lead to sudden cardiac death. We previously showed that a reduction in extracellular K(+) concentration ([K(+)](o)) prolongs QT intervals in intact rabbits, and decreases the cell surface density of I(Kr) in rabbit ventricular myocytes and of the HERG channel expressed in human embryonic kidney (HEK) cells.
The goal of the present study was to gain insights into the mechanisms for low [K(+)](o) induced reduction in HERG expression levels.
Using patch clamp, Western blot and confocal imaging methods, we demonstrated that at low [K(+)](o), the HERG channel entered a novel nonconducting state. Furthermore, this novel functional state triggered rapid internalization and degradation of the cell surface HERG channels. Thus, our data demonstrated for the first time a direct link between a gating state and the plasma membrane stability of an ion channel, HERG. Using HERG-permeant cations and site-directed mutagenesis, we identified the sites in the channel which are involved in the K(+)(o) dependence of HERG channels.
Extracellular K(+) is a prerequisite for HERG function and membrane stability.
