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Cardiovascular Research 2003 57(3):651-659; doi:10.1016/S0008-6363(02)00774-5
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Copyright © 2003, European Society of Cardiology

Dissociation of E-4031 from the HERG channel caused by mutations of an amino acid results in greater block at high stimulation frequency

Kuniaki Ishii*, Mirei Nagai, Masahiro Takahashi and Masao Endoh

Department of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan

* Corresponding author. Tel.: +81-23-628-5234; fax: +81-23-628-5235. kuishii{at}med.id.yamagata-u.ac.jp

Received 8 August 2002; accepted 25 October 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We have reported identification of the amino acid whose mutation reduces effects of quinidine on the HERG channel. Although the residue (isoleucine at 647) is not in the recently reported methanesulfonanilide binding site, a single concentration of E-4031 (10 µM) was less effective to I647 mutant channels than wild type HERG channel. We designed the present experiment to further investigate influence of mutations at 647 on the effects of methanesulfonanilides. Methods and results: HERG channels were expressed in Xenopus oocytes and their currents were measured by a two-microelectrode voltage clamp method. Of the two mutations initially studied (I647A and I647F), the I647F had a greater influence and differentially affected the effects of dofetilide and E-4031. The IC50 for dofetilide of the two mutant channels (I647A and I647F) was increased only 2-fold, but the IC50 for E-4031 was increased 6-fold (I647A) and 14-fold (I647F). Aromatic residues other than phenylalanine were then substituted for I647, and found to reduce the effects of E-4031. Whereas E-4031 dissociated from the mutant channels during rested state, dofetilide little dissociated. The mutant channels that showed recovery from E-4031 block were inhibited greater at 1 Hz than at 0.1 Hz. Conclusions: The present results indicate that dissociation of a drug from the HERG channel results in greater block at high frequency. Although the mechanism by which the mutations cause the dissociation of E-4031 is uncertain, it is noteworthy that one methanesulfonanilide dissociates from the channel more easily than another.

KEYWORDS Antiarrhythmic agents; Arrhythmia (mechanisms); K-channel; Long QT syndrome


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Methanesulfonanilide compounds such as dofetilide, E-4031 and MK-499 are potent and selective blockers of the IKr channel [1–3] that is encoded by the human ether-a-go-go-related gene (HERG) [4,5]. Blocking IKr by drugs results in prolongation of action potential duration (APD) and the excess in acquired long QT syndrome (LQTS) that can lead to the life-threatening ventricular tachyarrhythmia called torsades de pointes [6]. Unfortunately, currently available methanesulfonanilides all prolong APD more efficiently during bradycardia than during tachycardia [7,8]. This undesirable characteristic makes the compounds more prone to cause acquired LQTS. One reason for their larger effects on APD in bradycardia is that the block of IKr does not recover or recovers extremely slowly [9]. Recently, trapping of a methanesulfonanilide, MK-499, in the inner vestibule of the HERG channel by the activation gate was demonstrated using the unique HERG mutant that opens upon hyperpolarization [10]. When trapping occurs, the drug cannot exit from the channel as long as the activation gate is closed. This trapping mechanism is probably true for all the known methanesulfonanilides, and explains the lack of the rate dependence of IKr block by them. Rapid dissociation from the channel at diastolic potentials is a prerequisite for a drug to exhibit greater block at high frequency.

Molecular determinants of methanesulfonanilide binding have been studied using HERG channels expressed heterologously [11–14]. A phenylalanine at 656, which is located on the C-terminal half of the S6, was reported to be a molecular determinant of high-affinity dofetilide binding [13]. Subsequently, a more complete alanine-scanning mutagenesis study identified the methanesulfonanilide MK-499 binding site that is composed of six amino acids located on the S6 and the pore helix [14].

We did the experiments to search for amino acids that affect quinidine binding to the HERG channel, and have reported that mutations of isoleucine at 647 (I647) reduce the effects of various drugs including quinidine and E-4031 [15]. Interestingly, the homology model of the HERG channel, which was created by using the KcsA structure as the template [26], indicated that the residue faces toward the outside of the channel pore. The present experiment was carried out to further investigate how the mutations of I647 affect methanesulfonanilide bindings. Here we show that mutations of I647, especially with aromatic substitution, resulted in dissociation of E-4031 from the channels during rested state. In addition, E-4031 inhibited those mutant channels greater at a higher stimulation frequency.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Site-directed mutagenesis
The plasmid containing HERG cDNA (HERG/pGH19) was generously provided by Dr G.A. Robertson (University of Wisconsin, Madison, WI, USA) [5]. The origin of the pGH19 vector is the pGHE expression vector constructed by Liman et al. [16].

The procedure making point mutations at residue 647 in the S6 of HERG was described in detail elsewhere [15]. In brief, two unique restriction sites (a BglII site at the beginning of the S6 and an SphI site in the C-terminal region) were utilized to construct the mutants. Sense primers containing the desired mutations and an antisense primer were used for PCR reaction. The fragment amplified by PCR was digested with BglII and SphI, and ligated to HERG between the two restriction enzyme sites. In addition to point mutations, two amino-terminal deletion mutants (HERG WT{Delta}2-345 and I647F{Delta}2-345) were constructed by PCR. The procedure constructing the deletion mutants was also described in detail elsewhere [15]. Sequences of all the fragments generated by PCR were verified by the dideoxy chain termination method using a DNA sequencer (Model 310, Applied Biosystems, Foster City, CA, USA).

2.2 Expression and current recordings
Oocyte preparation and electrophysiological recording were carried out essentially as reported previously [17]. The pGH19 vectors containing the constructs were linearlized with NotI, and capped cRNAs were prepared from these templates with T7 RNA polymerase (Stratagene, La Jolla, CA, USA). Transcribed RNAs were dissolved in sterile water at a final concentration of 0.2–0.4 µg/µl for oocyte injection. The integrity of the cRNA products was checked by running the samples on agarose gels containing formaldehyde. Defolliculated Xenopus oocytes (stages V–VI) were injected with 45.6 nl of cRNA using a motor-driven injector (Drummond, Broomall, PA, USA). The injected oocytes were incubated in Barth's medium supplemented with penicillin G (71.5 units/ml) and streptomycin (35.9 µg/ml) at 18 °C for 2–6 days before electrophysiological measurements. The K+ currents were recorded by a conventional two-microelectrode voltage clamp method with 3 M KCl-filled electrodes. The bath recording solution consisted of ND 96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4), and all electrophysiological measurements were carried out at room temperature (21±1 °C). Current recordings were low pass-filtered at 1 kHz, analog-to-digital (A/D) sampled at 0.5 ms interval except in the long pulse protocol (Fig. 2A–C) where sampled at 13 ms interval. All data are expressed as the mean±S.E.M. The statistical significance was evaluated using Student's unpaired t-test. A P-value smaller than 0.05 was considered to be significant.


Figure 2
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  		Fig. 2 Time-dependent block by methanesulfonanilides. Representative current traces recorded in the absence and presence of 3 µM E-4031 (upper panels) and 3 µM dofetilide (lower panels) are shown (A, WT; B, I647F; C, I647A). Currents were elicited by 90-s depolarizing pulses to 0 mV from a holding potential of –80 mV. Ratios of current block induced by E-4031 (D) and dofetilide (E) were calculated and plotted against time after onset of the second pulse. Data were fit with a single exponential function (solid lines).

 
2.3 Drugs
Dofetilide, provided by Pfizer Central Research (Sandwich, Kent, UK), was dissolved in distilled water acidified with HCl to about pH 3.0 to make a 10 mM stock solution. E-4031, purchased from Wako Chemicals (Osaka, Japan), was dissolved in distilled water to make a 100 mM stock solution. Before use, both drugs were diluted with ND 96 solution (pH 7.4) to the desired concentration.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Mutations of isoleucine 647 reduce the sensitivity to methanesulfonanilides
Representative current traces of HERG WT, I647F and I647A channels and their corresponding current–voltage relationships are shown in Fig. 1A–C. Oocytes expressing these channels were held at –80 mV and depolarized to test potentials for 2 s with a return pulse to –60 mV. I647F channel current showed slower kinetics of deactivation, whereas the I647A channel current deactivated in a manner similar to HERG WT current. The current–voltage relationship for the I647F channel was shifted to the negative direction by about 10 mV. However, the voltage shift was unlikely to have influenced the effects of methanesulfonanilides, since inhibition of HERG WT current by the compounds was not voltage-dependent between –20 and +20 mV (data not shown). With respect to the voltage-dependence, there was no remarkable difference between the current–voltage relationships of I647A and WT channels. The effects of drugs on these channels were evaluated with the tail currents at –60 mV following a depolarizing test pulse to 0 mV. After confirming the stability of the control currents for at least 15 min, the oocytes were equilibrated with the drugs and pulsed repetitively every 5 min at 0.1 Hz (WT and I647A) or 0.05 Hz (I647F) until steady-state block was achieved. The I647F channel current was elicited at 0.05 Hz because of its slower deactivation. Concentration–inhibition curves for E-4031 are shown in Fig. 1D. Both I647A and I647F channels showed reduced sensitivity to E-4031, but the extent of the reduction was different. The IC50 values were 1.39±0.19 µM for WT (n=9), 8.10±1.21 µM for I647A (n=7) and 20.0±0.19 µM for I647F (n=7). In contrast, the effect of dofetilide was affected much less than E-4031. The IC50 values for dofetilide were 0.49±0.05 µM for WT (n=4), 0.86±0.08 µM for I647A (n=5) and 0.84±0.08 µM for I647F (n=4) (Fig. 1E).


Figure 1
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  		Fig. 1 Mutations of I647 reduced the affinity for methanesulfonanilides. In HERG WT (A), I647F (B) and I647A (C), currents were elicited by 2-s depolarizing pulses ranging from –70 mV to +40 mV in 10 mV increment. The holding potential was –80 mV and the repolarizing pulse was to –60 mV. Test pulses were applied every 10 s for WT and I647A, and every 20 s for I647F. Currents at the end of depolarizing pulses (open circle) and the peak of tail (closed circle) are plotted against membrane voltage. Concentration–inhibition curves for E-4031 (D) and dofetilide (E) determined by the tail currents following a step pulse to 0 mV are shown. Data were fit with a Hill equation: I/Icontrol=1–{1/(1+(IC50/D)n}, where I/Icontrol is relative current, IC50 is the concentration producing 50% inhibition, D is the drug concentration, and n is the Hill coefficient. The Hill coefficients of concentration–inhibition curves for dofetilide were 1.5 for WT, 1.3 for I647A and I647F, and those for E-4031 were 1.1 for WT, 1.0 for I647A, and 1.2 for I647F.

 
3.2 Time course of block by methanesulfonanilides
The time course of the blocks by E-4031 and dofetilide was assessed using prolonged depolarizing pulses. After control current was elicited by a 90-s pulse to 0 mV, the oocyte was equilibrated with the drug for 20 min at –80 mV and then the same test pulse was repeated. The current declined slowly as channel block by the drug developed but the initial phase of current activation was not affected, as shown in Fig. 2A–C (upper panels, E-4031; lower panels, dofetilide). The time course of changes in the ratio of current block is shown in Fig. 2D (E-4031) and 2E (dofetilide). The curves for E-4031 block were fitted by a single exponential function with time constants of 22.8±2.27 s for WT (n=7), 24.9±0.93 s for I647A (n=4) and 36.3±6.62 s for I647F (n=4). The current decay induced by E-4031 was slightly slower in the I647F channel than in the WT and I647A channels. From the exponential fit, the maximum inhibition in the I647A and I647F channels was significantly smaller than in the WT: 90.3±1.18% for WT, 81.8±1.23% for I647A (P<0.001 vs. WT) and 76.3±2.89% for I647F (P<0.001 vs. WT). In contrast, the maximum inhibition induced by dofetilide was not suppressed in the mutants: 87.8±3.84% for WT, 95.2±0.93% for I647A and 91.1±2.26% for I647F. The time constants of the current decay induced by dofetilide were 17.5±1.55 s for WT (n=4), 18.2±4.37 s for I647A (n=4) and 25.3±3.27 s for I647F (n=5).

3.3 E-4031 dissociates from I647A and I647F
Dissociation of E-4031 and dofetilide from the channels during rested state was assessed (Fig. 3). Test pulses to 0 mV (400 ms) followed by a return pulse to –60 mV (200 ms) were repetitively applied at 1 Hz. When the current became stable for at least 10 min, the oocyte was held at –80 mV for varying periods and then the same test pulse was applied to see whether the current is stable after the –80 mV holding. If the oocytes showed unstable currents with the holding periods at –80 mV, such cells were discarded. In the presence of E-4031 (3 µM for WT, 10 µM for I647A and I647F) or dofetilide (1 µM for WT, I647A and I647F), the same protocol was applied to assess the recovery from the block. Effects of the drugs were evaluated with the tail currents at –60 mV. E-4031 inhibited WT current by 81.1±4.63%, I647A current by 63.7±4.05% and I647F current by 61.2±5.86%. Dofetilide inhibited WT current by 91.2±1.25%, I647A current by 82.9±4.44% and I647F current by 79.1±5.80%. The percentage recovery from E-4031 block plotted against time is shown in Fig. 3A. The HERG WT current did not recover from E-4031 block, whereas I647F channel current showed remarkable recovery (16.1±1.5% recovery at 30 s and 20.8±2.3% at 1 min, n=6), which indicates dissociation of E-4031 from the I647F channel during rested state. In the I647A channel, small but obvious recovery was observed (4.3±0.7% at 1 min, n=7, Fig. 3A). In the case of dofetilide, the HERG WT current hardly recovered from the block, which was similar to E-4031, and I647A and I647F channel currents recovered only slightly (about 4% at 1 min, Fig. 3B).


Figure 3
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  		Fig. 3 Differential dissociation of E-4031 and dofetilide from I647A and I647F. Voltage pulse protocol is shown in top. Xenopus oocytes expressing the HERG channels were continuously stimulated by the 400-ms test pulse to 0 mV at 1 Hz for the control and until the inhibition by the drug reached steady state. The return pulse was to –60 mV. After that the oocytes were held at –80 mV for varying periods up to 1 min in the continuous presence of the drug, and then the current was elicited by the same test pulse to evaluate the recovery from the block. The percentage recovery from the block by E-4031 (A) and dofetilide (B) is plotted against the time held at –80 mV after the steady-state block. * P<0.05, ** P<0.01, *** P<0.001 vs. WT.

 
3.4 Influence of aromatic substitution for I647 on E-4031 binding
Since I647F had larger effects on E-4031 binding than I647A, we studied the influence of other aromatic substitutions for I647. As shown in Fig. 4, Tyr substitution reduced the effect of E-4031 (10 µM) to similar extent as Phe substitution: I647F was inhibited by 25.3±3.48% (n=7) and I647Y was inhibited by 25.9±7.11% (n=4). In contrast, Trp substitution reduced the effect of E-4031 (10 µM) significantly, but to much less extent than Phe substitution: WT channel was inhibited by 88.6±1.07% (n=8) and I647W was inhibited by 55.8±6.05% (n=10; P<0.001 vs. WT). We next assessed the recovery from E-4031 block of I647Y and I647W. However, we could not estimate the current recovery of I647Y channel since the currents of this mutant were unstable after the period of –80 mV holding. As shown in Fig. 5A, I647W currents recovered from E-4031 block slightly less than I647F.


Figure 4
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  		Fig. 4 Reduced effects of E-4031 on I647Y and I647W. (A) Current traces of WT, I647F, I647Y and I647W are shown in the left column. Currents were elicited by 2-s depolarizing pulses ranging from –70 to +40 mV in 10 mV increment. In the right column, currents at the end of depolarizing pulses (open circle) and the peak of tail (closed circle) are plotted against membrane voltage. (B) Effects of 10 µM E-4031 on the currents were evaluated with the tail currents following a voltage step to 0 mV. *** P<0.001 vs. WT.

 

Figure 5
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  		Fig. 5 Frequency-dependent block by E-4031. (A) Recovery from the block by E-4031 of I647W was assessed similarly as described in the legend of Fig. 3. The percentage recovery from the block by E-4031 is plotted against the time held at –80 mV after the steady-state block. The data on WT and I647F are the same as in Fig. 3. *** P<0.001 vs. WT. (B–D) Currents were elicited by 400-ms depolarizing pulse to +20 mV at frequencies of 1 Hz (closed symbol) and 0.1 Hz (open symbol). In the presence of E-4031 (3 µM for WT, 10 µM for I647F and I647W), tail currents at –60 mV relative to the control (before the drug application) currents are plotted against time after the first pulse. Data were fit with a single exponential function.

 
3.5 Frequency-dependent block
We then investigated whether difference in stimulation frequency affects E-4031 block of WT, I647F, and I647W channels. Depolarizing pulses to +20 mV for 400 ms from a holding potential of –80 mV were applied repetitively at 1 and 0.1 Hz. A return pulse was to –60 mV where the tail current was used to evaluate the effect of E-4031. In Fig. 5B–D, relative currents are plotted against time after the end of wash-in of E-4031. From the fitting of data with single exponential function, E-4031 appeared to inhibit WT currents to 0.172±0.023 at 0.1 Hz (n=4) and to 0.176±0.001 at 1 Hz (n=6). The time constants for E-4031 block of WT were 850.9±48.35 s at 0.1 Hz and 87.6±0.56 s at 1 Hz. Thus WT currents were inhibited by E-4031 faster at the higher stimulation frequency, but the steady-state level of the block was similar at the both frequencies. In contrast, I647F and I647W currents were inhibited by E-4031 greater and faster at the higher stimulation frequency. At 0.1 Hz, I647F and I647W currents were inhibited to 0.686±0.001 (n=9) and 0.580±0.004 (n=7), respectively, whereas at 1 Hz they were inhibited to 0.400±0.001 (n=6) and 0.230±0.001 (n=5), respectively. The time constants for E-4031 block of I647F currents were 97.5±3.44 s at 0.1 Hz and 34.9±0.33 s at 1 Hz, and those of I647W were 165.2±6.37 s at 0.1 Hz and 33.9±0.41 s at 1 Hz.

3.6 Restoration of deactivation in I647F does not restore the effects of E-4031
Since I647F channel current deactivated more slowly than WT (Fig. 1A and B), we investigated whether the change in deactivation kinetics itself contributes to the reduced effects of E-4031 on the I647F channel. It has been reported that the amino-terminus regulates deactivation of HERG current and deletion of the domain results in acceleration of deactivation [18]. Therefore, the amino-terminal deletion mutant of I647F (I647F{Delta}2-354) was constructed to cancel the slowing of deactivation (Fig. 6B). When the decaying phase of the tail current at –80 mV was fitted with a double exponential function, the time constant of the rapid component was 187.5±9.2 ms for WT, 437.1±23.2 ms for I647F and 75.0±4.0 ms for I647F{Delta}2-354 (n=5 for each). Fig. 6C shows the effects of 10 µM E-4031 on the currents of the WT, I647F and I647F{Delta}2-354 channels. Whereas HERG WT current was inhibited by 89.1±0.74%, the current of I647F{Delta}2-354 was inhibited by 47.3±6.16%.


Figure 6
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  		Fig. 6 Effects of E-4031 on the mutant with the amino-terminal deleted. Current traces of WT and WT{Delta}2-354 are shown in (A), and those of I647F and I647F{Delta}2-354 are shown in (B). Currents were elicited by 2-s depolarizing pulses ranging from –70 to +40 mV in 10 mV increment. The return pulse was to –60 mV. (C) Effects of 10 µM E-4031 on the currents evaluated with the tail currents following a voltage step to 0 mV are shown. The inhibition of the current of I647F was 31.3±2.58%, which was slightly but significantly smaller than that of I647F{Delta}2-354 (47.3±6.16%). *** P<0.001 vs. WT.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We have previously identified the isoleucine at 647 of the HERG channel as the residue whose mutation affects the binding of quinidine [15]. The residue was not in the recently identified methanesulfonanilide binding site [14]. However mutations of I647 reduced sensitivity not only to quinidine but also to other drugs including E-4031. The present study was designed to further investigate the influence of I647 mutations on methanesulfonanilide bindings. I647A and I647F channels were subjected to increasing concentrations of E-4031 and dofetilide, which showed that I647F had a greater influence than I647A on E-4031 block, whereas both had similar effects on dofetilide block. There are two factors that might be responsible for the less effect of E-4031 on the I647F channel than on WT: facilitation of drug dissociation from the rested state channel and decreased affinity to the depolarized channel (open and inactivated channel). During 10 s of rested state, approximately 10% of E-4031 dissociated from the I647F channel, and during 20 s about 15% dissociated. To construct concentration–inhibition curves, a test pulse was applied every 20 s to the I647F channel because of its slower deactivation. Thus, it is likely that dissociation of the drug contributed to the observed reduction of the E-4031 block. In addition to the dissociation from the rested state channel, the binding of E-4031 to the depolarized channel seemed to be affected by the I647F mutation. The time course of E-4031 block of the I647F channel was slower and the maximum inhibition was less than in the WT channel as shown in the long pulse experiment (Fig. 2). The two factors must have contributed to the less effect of E-4031 on the I647F. In the case of the I647A, dissociation of E-4031 from the rested state channel was much less than in the case of I647F and the binding to the depolarized channel was affected less. These data are in good agreement with the lower influence of the I647A mutation on the effects of E-4031.

MK-499 is the only methanesulfonanilide for which direct evidence of trapping has been demonstrated, but other methanesulfonanilide compounds are also thought to block HERG channels by a similar trapping mechanism. Block of IKr/HERG by methanesulfonanilides requires opening the channel and recovery from the block is reported to be very slow or irreversible [19–24]. We also observed that block of the HERG channel by E-4031 barely recovered even after a 20 min washout (data not shown). Therefore, it is likely that most methanesulfonanilides are trapped in the inner vestibule between the selectivity filter and the activation gate. Trapped compounds cannot dissociate from the channel during rested state. Our results indicate that mutations of I647 lead to dissociation of methanesulfonanilide compounds, especially E-4031. One possible mechanism that causes the dissociation is disruption of the activation gate by the mutation. Another possibility for the dissociation is that the mutations narrowed the inner vestibule in such a manner that the mutants are unable to accommodate the drug efficiently. In the Shaker K+ channel, the opposite (widening of the inner vestibule by mutation) has been reported. The Shaker K+ channel is blocked by internally applied TEA but it cannot trap this compound [25]. However, with a point mutation in the S6 the channel becomes able to trap TEA and also even a larger compound, decyltriethylammonium [25]. We have reported that the side chain of amino acid at 647 extended toward the outer helix of the S5 [15]. Although the mechanism by which the mutations at 647 cause the dissociation of a methanesulfonanilide is unclear at present, I647F and I647W channels recovered from the E-4031 block greater than I647A channel. It might be possible that a bulky residue, such as Phe and Trp at 647 causes shift of the S6 toward the inside to narrow the inner vestibule of the HERG channel pore.

Since the I647F mutation caused a slowing of deactivation kinetics, the possible influence of the slow deactivation on the drug binding was evaluated with an amino-terminal deletion mutant of I647F (I647F{Delta}2-354) in which the slow deactivation was reversed. The effects of E-4031 on I647F was slightly smaller than those on I647F{Delta}2-354, which might be explained by dissociation of E-4031 during deactivation. Alternatively, the amino-terminal domain might have some influence on the binding of E-4031. However, since I647F{Delta}2-354, which deactivated even faster than the WT, was less inhibited by E-4031, it is evident that mutations at 647 influence the affinity for E-4031 besides changing deactivation kinetics.

In summary, the present study demonstrates that the mutations at I647 of the HERG channel cause dissociation of a methanesulfonanilide, E-4031. The dissociation results in greater block of the mutant channel by E-4031 at higher frequency. If an antiarrhythmic drug blocks IKr with slow onset and allows recovery from the block due to dissociation during the rested state, the drug might prolong APD more efficiently during tachycardia than during bradycardia. In this regard, it is noteworthy that the two methanesulfonanilides (E-4031 and dofetilide) dissociated from the mutant channel to different extents. Preliminary data in our ongoing study suggest that a novel methanesulfonanilide can dissociate from the HERG WT channel. Further studies using the I647 mutants and various methanesulfonanilides might be able to correlate molecular properties of the compounds with their trapping in the inner vestibule of the channel.

Time for primary review 22 days.


   Acknowledgements
 
This study was supported by Grants-in Aid for Scientific Research (No. 10470021 and 12670081) from the Ministry of Education, Science, Sports and Culture, Japan, The Naito Foundation, and The Mochida Memorial Foundation for Medical and Pharmaceutical Research. We thank Dr Gail Robertson for providing HERG/pGH19 construct. We also thank Drs K. Ono and K. Ito for helpful discussion, and Dr K. Nunoki for review of the manuscript. Dofetilide was kindly provided by Pfizer Central Research.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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