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Vol. 60, Issue 6, 1343-1348, December 2001
Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, School of Medicine, Cleveland, Ohio (E.F., A.M.B.); and I. Physiologisches Institut, Universitaet Heidelberg, Heidelberg, Germany (W.J.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The major subunit of the cardiac delayed rectifier current IKr is encoded by the human ether a-go-go related gene (HERG). HERG/IKr channels are blocked selectively by class III antiarrhythmic methanesulfonanilide drugs such as dofetilide. The binding site for methanesulfonanilides is believed to be similar for nonantiarrhythmic drugs such as antihistamines, antibiotics, and antipsychotics. To gain further insight into the binding site, we examined the minimal structural changes necessary to transform low-affinity binding of dofetilide by the related bovine ether a-go-go channel bEAG to high-affinity binding of HERG. Previously, it was shown that high-affinity binding in HERG required intact C-type inactivation; the bovine ether a-go-go K+ channel (bEAG), unlike HERG, is noninactivating. Therefore, we introduced C-type inactivation into noninactivating bEAG using site-directed mutagenesis. Two point mutations in the pore region, T432S and A443S, were sufficient to produce C-type inactivation. Low concentrations of dofetilide produced block of bEAG T432S/A443S; unlike HERG, block was almost irreversible. Substitution of an additional amino acid in transmembrane domain S6 made the block reversible. Dofetilide blocked the triply mutated bEAG T432S/A443S/A453S with an IC50 value of 1.1 µM. The blocking potency was 30-fold greater than bEAG WT and about one third that of HERG WT. We conclude that high affinity methanesulfonanilide binding to HERG channels is strongly dependent on C-type inactivation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The hereditary long QT
syndrome (LQTS) is caused by mutations in five known genes, four of
which encode potassium channel subunits (Keating and Sanguinetti,
2001
). Two gene products, KvLQT1 and minK produce 
and 
subunits
of the slowly activating cardiac delayed-rectifier potassium current
IKs. (Wang et al., 1996a; Splawski et al.,
1997). The human ether-a-go-go related gene HERG (Warmke and
Ganetzky, 1994) encodes the major subunit of the rapidly activating
cardiac delayed-rectifier potassium channel (Sanguinetti et al., 1995).
Mutations in HERG cause chromosome 7-linked LQTS (Curran et al., 1995)
and link structural changes in delayed rectifying potassium channels to
prolongation of the cardiac action potential, of the QT interval, and
of electrocardiogram readings.
HERG K+ channels have unique pharmacological
properties and are blocked with high affinity and selectivity by Class
III antiarrhythmic methanesulfonanilides, such as dofetilide, MK-499,
and E4031 (Jurkiewicz and Sanguinetti, 1993; Trudeau et al., 1995;
Kiehn et al., 1996; Snyders and Chaudhary, 1996; Spector et al., 1996).
The binding site that has been proposed (Lees-Miller et al., 2000;
Mitcheson et al., 2000b) makes HERG K+ channels a
major target for block by nonantiarrhythmic drugs, including the
antihistamines terfenadine (e.g., Roy et al., 1996) and astemizole
(Zhou et al., 1999), and the gastrointestinal prokinetic drug cisapride
(e.g., Rampe et al., 1997), that cause drug-induced LQTS as an unwanted
side effect.
Previous structure-function studies of high-affinity drug binding in
HERG K+ channels provided evidence that intact
C-type inactivation is crucial for drug binding, because many mutations
that disrupted inactivation dramatically reduced the sensitivity to
methanesulfonanilide drugs, most probably because of allosteric changes
induced in the drug binding site (Wang et al., 1997b; Ficker et al.,
1998; Herzberg et al., 1998; Lees-Miller et al., 2000). Similarly, it has been demonstrated that high-affinity drug binding can be modulated by extracellular cations such as K+,
Na+, or Cd2+, all of which
modify inactivation gating of HERG K+ channels
(Wang et al., 1997; Numaguchi et al., 2000).
Major progress toward a structural basis of high-affinity drug binding
has recently been made with the identification of two crucial aromatic
amino acid residues in the S6 transmembrane domain of the channel
protein: HERG Y652 and F656. These two amino acids seem to constitute a
major part of the methanesulfonanilide binding site in HERG with
additional contributions being made by residue G648 in S6 and residues
T623 and V625 in the pore helix of the HERG channel protein
(Lees-Miller et al., 2000; Mitcheson et al., 2000b). These residues
face the conduction pathway, and are accessible only in the open state
and drug molecules are trapped inside the conduction pathway by closure
of the activation gate (Mitcheson et al., 2000a,b).
With localization of the receptor for high-affinity drug binding to the
S6 transmembrane domain, the role played by C-type inactivation in
enhancing drug binding is problematic for the following reasons:
1) mutations such as HERG S620T, in which inactivation has been
completely removed, showed dramatically lowered drug-sensitivity despite the availability of F656 and Y652 (Ficker et al., 1998); 2) all
residues important for drug binding are conserved in the closely
related, noninactivating EAG K+ channels yet EAG
channels are relatively insensitive to block by dofetilide or MK-499
(Ficker et al., 1998; Herzberg et al., 1998; Mitcheson et al., 2000b);
3) mutations at several of the binding loci (e.g., HERG G648A, T623A,
F656A) showed a large negative shift in inactivation (i.e., increased
inactivation); contrary to expectations, however, they were less
sensitive to drug block than WT channels (Mitcheson et al., 2000b); and
4) some mutations that completely removed inactivation remained rather
sensitive to methanesulfonanilide block (e.g., HERG S620C or HERG
G628C/S631C; Wang et al., 1997; Ficker et al., 1998; Mitcheson et al.,
2000b). These inconsistencies show that the interaction between
inactivation and the pore lining residues implicated in drug binding is
not understood.
In the present experiments, we examined this interaction by addressing the difference in drug sensitivity between HERG and EAG channels. We mutated the noninactivating EAG family member bEAG, which is about 100-fold less sensitive to dofetilide than HERG, by substitution of two amino acids at positions 432 and 443 in the pore region. These mutations introduced HERG-like C-type inactivation and high-affinity dofetilide binding simultaneously. However, block by dofetilide was almost irreversible, even at low concentrations, unlike the situation in HERG. To convert bEAG T432S/A443S into a channel that was blocked reversibly by dofetilide, we mutated one additional residue in the S6 transmembrane helix. Taken together, our results show that introducing C-type inactivation into bEAG was sufficient to transform the low affinity methanesulfonanilide binding site of bEAG into a high-affinity site resembling HERG channels.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of Mutant Channels.
HERG WT cDNA was a gift
from Dr. M. T. Keating (University of Utah, Salt Lake City, UT).
bEAG WT cDNA was provided by Dr. A. Baumann (Forschungszentrum Juelich,
Juelich, Germany). All point mutations in HERG and bEAG were
generated by overlap extension polymerase chain reactions using
polymerase chain reaction-generated MluI-KpnI
cassettes anchored in pBluescript as template (Ficker et al., 1998).
Before subcloning, the cassettes were sequenced. cRNA was prepared
using the mMessage mMachine in vitro transcription kit (Ambion, Austin,
TX) and SP6 polymerase after linearization with EcoRI (HERG
WT, and point mutations in HERG) or EcoRV (bEAG WT, and
point mutations in bEAG).
Electrophysiology.
Isolation, maintenance, and injection of
Xenopus laevis oocytes were performed as described
previously (Ficker et al., 1998). Whole-cell currents were recorded 2 to 7 days after cRNA injection using standard two-microelectrode
voltage clamp techniques. Bath solutions were 96 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES (5K Ringer, pH 7.4) and 1.8 mM
CaCl2, 10 mM HEPES with either 115 mM KCl (115K
Ringer, pH 7.4), 115 mM RbCl (115Rb Ringer, pH 7.4), 115 mM CsCl (115Cs
Ringer, pH 7.4), or 115 mM NaCl (115Na Ringer, nominally
K+-free, pH 7.4). Bath solutions containing 100 mM [K]ex, 30 mM [K]ex,
and 5 mM [K]ex with 30 mM TEA added were
prepared by equivalent reductions in the concentration of NaCl in 5K
Ringer or by omission of KCl for a nominally
K+-free 100Na Ringer. For
IC50 measurements, dofetilide was perfused in
increasing concentrations with 5K Ringer. Dofetilide was provided by
Pfizer Central Research (Groton, CT). All other chemicals were obtained from Sigma (St. Louis, MO). Macropatch recordings were performed using an EPC-7 patch clamp amplifier (List, Darmstadt, Germany). Patch pipettes had resistances of 0.2 to 0.6 M
and were
filled with 5K Ringer (see above). For patch recordings the bath
solution had the following composition: 100 mM KCl, 5 mM EDTA, 5 mM
EGTA, 10 mM HEPES (isoK, pH 7.4). No leak subtraction was applied. All
recordings were performed at room temperature (20-22°C). pClamp
software (Axon Instruments, Foster City, CA) was used for the
generation of voltage clamp pulses and for data acquisition. When
appropriate, data were expressed as mean ± S.E.M.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pore Mutations That Introduce C-Type Inactivation in bEAG.
Several reports have shown that amino acid residues in positions 620 and 631 are crucial for C-type inactivation in HERG channels (Schoenherr and Heinemann, 1996; Smith et al., 1996; Ficker et al.,
1998; Herzberg et al., 1998). To introduce C-type inactivation in bEAG,
we placed serines at position 432 or 443 of bEAG because these
positions are equivalent to S620 and S631 in HERG. Neither bEAG A443S
nor bEAG T432S introduced the inactivating current phenotype that was
desired (Ficker et al., 1998).
20, 0, and +20 mV,
respectively. These values were about double the time constants
measured in macropatch recordings of HERG WT. In HERG, time constants
were 12.4 ± 0.6, 10.6 ± 0.5, and 7.4 ± 0.6 ms at
20, 0, and +20 mV, respectively (n = 6). The
difference in time constants may be related to the different voltage
protocols that were used. In bEAG T432S/A443S, we analyzed current
inactivation using depolarizing voltage commands at which channel
activation and inactivation proceeded simultaneously (Fig. 1A). In
HERG, a three-step pulse protocol was used that isolated inactivation
from activation (Smith et al., 1996). For similar reasons, fractional
inactivation differed between the two channels. At more hyperpolarized
membrane potentials, at which current activation was slow, bEAG
T432S/A443S currents seemed to inactivate much less than HERG WT
currents. At more depolarizing potentials, however, at which activation
was considerably faster, fractional inactivation approached the values
measured for HERG WT channels. In marked contrast, the inactivation
process in HERG S631A was shifted to more positive values (Fig. 1D; see
Zou et al., 1998). In addition, the time course for recovery from
inactivation was equally fast in bEAG T432S/A443S and HERG WT. At 80
mV, the time constant for recovery from inactivation was 17.1 ± 2.1 ms (n = 4; Fig. 1E) and is comparable with the time
constant of about 10 ms measured at 80 mV for HERG WT (Sanguinetti et
al., 1995).
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; Lopez-Barneo et al., 1993; Schoenherr
and Heinemann, 1996). TEA at 30 mM blocked bEAG channels by about 50%
and produced the expected slowing of inactivation over a wide range of
potentials (Fig. 2, A and B). Likewise,
inactivation was slowed by elevated
[K+]ex that at the same
time increased current amplitude (Fig. 2, C and D). Moreover, in high
K+, it was apparent that the tail currents of
bEAG T432S/A443S no longer deactivated almost instantaneously.
Deactivation of tail currents was slowed sufficiently that recovery
from inactivation produced a clearly resolved rising phase in inward
tail currents that in HERG WT channels was attributed to recovery from
C-type inactivation. In many potassium channels slowing of C-type
inactivation by extracellular cations followed the selectivity sequence
for permeation, namely
K+~Rb+ > Cs+ > Na+ (Lopez-Barneo et
al., 1993). We found that inactivation in bEAG T432S/A443S was slowed
most with Rb+ and Cs+
following the sequence Rb+ 
Cs+> K+ > Na+ (Fig. 2, E and F). As for HERG channels,
inactivation in bEAG T432S/A443S was strongly voltage-dependent (Wang
et al., 1996b).
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Dofetilide Block of bEAG T432S/A443S.
Intact C-type
inactivation has been shown to be a prerequisite for high-affinity
binding of methanesulfonanilide drugs in HERG K+
channels (Wang et al., 1997; Ficker et al., 1998; Herzberg et al.,
1998). Consequently, single point mutations at bEAG 432 or bEAG 443 that express noninactivating currents failed to produce high-affinity
drug binding in bEAG. BEAG T432S resulted in a channel only 4-fold more
sensitive to dofetilide than bEAG WT, whereas bEAG A443S currents were
slightly less sensitive than WT currents (Table
1).
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). Trapping of dofetilide in bEAG
T432S/A443S channels could be tested directly by the introduction of
additional mutations that either slow deactivation or induce reopenings
at negative membrane potentials as described for HERG D540K
(Sanguinetti and Xu, 1999). Along these lines, we described previously
a HERG/EAG chimera with bEAG S6 transplanted into HERG background
(HBS6; Ficker et al., 1998) that expressed a nonfunctional, permanently
opened activation gate combined with fully preserved C-type
inactivation. HBS6 channels were more sensitive to block by dofetilide
than HERG WT channels with drug block being reversible.
To quantify the increased sensitivity of bEAG channels to dofetilide
upon introduction of C-type inactivation, in the present study, we
engineered additional mutations in the bEAG T432S/A443S background to
achieve reversible blockade and measure half-maximal blocking
concentrations. We focused on two positions in the N-terminal half of
S6 at which hydrophilic S641 and C643 residues of HERG were represented
by hydrophobic A453 and A455 residues in bEAG. bEAG T432S/A443S+A455C
showed the behavior of bEAG T432S/A443S with respect to kinetics,
C-type inactivation, and irreversible dofetilide binding (data not
shown). In contrast, bEAG T432S/A443S+A453S activated slowly at
potentials more negative than 20 mV and exhibited no detectable
inactivation. At potentials more depolarized than 20 mV, however,
channels inactivated and produced a crossover of raw current traces
(Fig. 4A). The current-voltage relationship was bell-shaped (Fig. 4B)
and voltage-dependent inactivation was apparent when the holding
potential was shifted from 80 to 60 mV to accelerate current
activation (Fig. 4A, inset). Onset of dofetilide block was slow, but in
contrast to bEAG T432S/A443S, a steady state was reached within several
minutes. The block was now reversible; Fig. 4D shows the washout after
the application of 10 µM dofetilide, which blocked currents by about
90%. The time course of recovery from block could be approximated by
monoexponential functions with a mean time constant of 673 ± 53 s (n = 7). On average, 72 ± 6% of
control currents were recovered (n = 8). The washout
kinetics of bEAG T432S/A443S/A453S were considerably slower than the
time constant of 99 ± 9.8 s measured in bEAG WT after application of 100 µM dofetilide that blocked bEAG WT by about 90%
(n = 11). The recovery time constant of HERG-WT was
1450 ± 226 s (with 10 µM dofetilide) and was about double
the time constant of bEAG T432S/A443S/A453S. For comparison, the
extremely slow wash out kinetics of bEAG T432S/A443S is given in Fig.
4D. The differences in rates of recovery from 90% drug block in bEAG
WT, bEAG T432S/A443S/A453S, and HERG WT correlated with differences in
IC50 values. In bEAG T432S/A443S/A453S, the
IC50 value of dofetilide block was 1.1 ± 0.2 µM (n = 7, Fig. 4C and Table 1). This channel construct was about 30-fold more sensitive to dofetilide than bEAG WT
(IC50, 31.8 ± 7.5 µM, n = 6) and only about 3-fold less sensitive than HERG WT
(IC50, 0.32 ± 0.04 µM, n = 24).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mapping the binding site for dofetilide in HERG channels is complicated by tight coupling between binding and C-type inactivation. To complement experiments done with "loss of function" mutations in HERG, we adopted a "gain of function" strategy in bEAG and showed that mutation of two amino acid residues in the pore region introduced C-type inactivation and high-affinity dofetilide binding.
Successful transfer of C-type inactivation into bEAG relied upon the
analysis of structural inactivation determinants in HERG. In general,
C-type inactivation is sensitive to mutations in or close to the pore
region (e.g., Hoshi et al., 1991; Lopez-Barneo et al., 1993). C-type
inactivation in HERG was altered by mutating position 631, a pore
residue homologous to Shaker 449 in the external mouth of the pore
(Schoenherr and Heinemann, 1996). Mutation of HERG S620 to T620, a
residue located at the inner end of the pore helix and not exposed to
the conduction pathway as judged from Doyle et al. (1998) had even more
pronounced effects, completely abolishing C-type inactivation (Ficker
et al., 1998; Herzberg et al., 1998). Interestingly, HERG S620 is
homologous to position 369 in Kv2.1, a residue with large effects on
current inactivation (DeBiasi et al., 1993). These results raise the
question of how residues facing opposite sites of the membrane are
involved in C-type inactivation. HERG S631 localizes to the external
mouth of the pore and by analogy with Shaker 449 is believed to control access of external ions to a more internally located C-type
inactivation site (Molina et al., 1997). HERG S620 might affect C-type
inactivation by contributing to ion occupancy at a critical site in the
selectivity filter as demonstrated for Shaker A463C (Ogielska and
Aldrich, 1999). The proposition that both HERG620 and HERG631 regulate ion occupancy at the C-type inactivation site (Herzberg et at., 1998),
is further supported by our results with reverse mutations in bEAG.
Neither bEAG T432S nor bEAG A443S alone expressed C-type inactivating
currents, whereas the combination of both mutations in bEAG T432S/A443S
introduced an inactivation process with the hallmarks of C-type inactivation.
Given the tight coupling between C-type inactivation and high-affinity
drug binding in HERG, our results showed, not unexpectedly, that C-type
inactivating bEAG T432S/A443S and bEAG T432S/A443S/A543S channels were
blocked by low concentrations of dofetilide. Previous work in Shaker
K+ channels suggested that conformational changes
of C-type inactivation were restricted to the selectivity filter (Liu
et al., 1996; Molina et al., 1997; Harris et al., 1998). By contrast,
critical structural determinants for high-affinity drug binding in HERG
have been located to positions 652 and 656 in the S6 transmembrane
domain, positions that are conserved in EAG channels (Lees-Miller et
al., 2000; Mitcheson et al., 2000). How, then, can C-type inactivation communicate with a drug-binding site controlled by S6 residues facing
the internal vestibule and conduction pathway? One possibility is that
C-type inactivation interacts with a rotational movement of S6 during
activation making crucial residues in S6 accessible for drug binding.
In the closed state of the HERG channel, these residues are hidden
consistent with the absence of resting block by methanesulfonanilides
(Kiehn et al., 1996; Snyders and Chaudhary, 1996). In Shaker channels,
it has been shown that small changes in the size of a side chain
(Shaker I470C) allow blockers suddenly to become trapped in the closed
state of the channel protein; it has been suggested that structural
differences between channels that do and do not trap blockers are only
minor (Holmgren et al., 1997). Therefore, it is conceivable that point
mutations affecting C-type inactivation may change the size of the
internal vestibule of channels in the EAG gene family and thereby allow
or impede trapping of methanesulfonanilides. Because trapping of
high-affinity blockers has important consequences for the reversibility
of block in HERG channels leading to accumulation of drug effects, it
may be important in future experiments to determine the relationship between inner vestibule size, C-type inactivation, and drug binding more precisely to develop new blocking molecules that might escape from
their binding sites more readily.
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Acknowledgments |
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We thank Dr. M. T. Keating for the HERG cDNA clone; Dr A. Baumann for the bEAG cDNA clone; T. Carroll and Dr. W.-Q. Dong for expert technical assistance; Dr. B. Wible for help with some of the mutations; and Dr. G. Kirsch for helpful comments on the manuscript.
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Footnotes |
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Received June 13, 2001; Accepted September 7, 2001
1 Present address: Merck Sharp and Dohme Research Laboratories, Neuroscience Research Center, Harlow, Essex, UK.
This study was supported by a Grant-in-aid award from the American Heart Association, Northeast Ohio Affiliate, Inc (to E.F.); and by National Institutes of Health Grants HL61642, HL36930 and DK54178 (to A.M.B.).
Dr. Eckhard Ficker, Rammelkamp Center, MetroHealth Medical Center, 2500 MetroHealth Drive, Cleveland, OH 44109-1998. E-mail: eficker{at}metrohealth.org
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Abbreviations |
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LQTS, long QT syndrome; IKr, cardiac delayed rectifier; dofetilide, N-[4-(-{-[4-(methanesulfonamino)-phenoxyl]-N-methylethylamino}ethyl)phenyl]methanesulfonamide; HERG, human ether a-go-go related gene; MK-499, (+)-N-[1'-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4'-piperidin)-6-yl]methanesulfonamide] monohydrochloride; bEAG, bovine ether a-go-go K+ channel; WT, wild-type; TEA, tetraethylammonium.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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), HERG S631A (
), and bEAG
T432S/A443S (
), n = 5 to 7. Fractional
inactivation was calculated in two-microelectrode recordings from peak
and steady-state currents elicited with pulse protocols as shown in A
for bEAG T432S/A443S. For HERG WT and HERG S631A, fractional
inactivation was analyzed using instantaneous current-voltage protocols
with test pulses following directly on 25- or 4-ms steps to 
); n = 5 to 8. E, effects
of 115K+, Rb+, Cs+, and
Na+ on current inactivation. F, time constants with
different monovalent cations in bath solution: 115K (
) by 1 µM
dofetilide. Only partial washout could be obtained. For comparison,
time-dependent dofetilide block of bEAG WT (
) and HERG WT (
) is
shown. In contrast to bEAG T432S/A443S, 80 to 100% of control values
could be recovered in HERG WT during washout.
