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Vol. 57, Issue 2, 367-374, February 2000
Department of Medicine, University of Calgary, Calgary, Alberta, Canada.
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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This study reports that the affinity of HERG1 A for dofetilide is decreased from 0.125 ± 0.003 µM for wild-type (WT) channels to 15 ± 3 µM for F656V, a mutation in the COOH-terminal half of the S6. Similarly, the IC50 for quinidine was increased from 8 ± 4 µM for WT to 219 ± 65 µM for the F656V mutation, whereas affinity for external tetraethylammonium was similar for WT (51 ± 10 mM) and F656V (36 ± 10 mM, NS). Kinetics of onset of inactivation of F656V was similar to WT but kinetics of deactivation, activation, and recovery from inactivation differed from WT. However, mutations in nearby amino acids in the S6 more strikingly altered deactivation, activation, and recovery from inactivation but had little effect on affinity for dofetilide. To assess the effects of disruption of inactivation, the S631A mutation was made. The S631A mutation altered the IC50 for dofetilide to 20 ± 3 µM, but the IC50 for quinidine was unchanged at 8 ± 4 µM for WT and 10 ± 1 µM for S631A. To address whether the F656V mutation alters the IC50 for dofetilide in a channel that does not inactivate, the double mutation S631A/F656V was made. The IC50 for dofetilide of the double mutation was 32 ± 3 µM, which is not substantially different than that of S631A. These data support the notion that allosteric changes occurring during the process of inactivation are necessary for high-affinity dofetilide binding. In conclusion, the Phe-656 residue of HERG is a molecular determinant of high-affinity dofetilide binding.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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HERG1
is a member of the ether a-go-go (EAG) family of genes that
encode voltage-gated potassium channels (Warmke and Ganetzky, 1994
;
Curran et al., 1995; Shi et al., 1997; Splawski et al., 1998). HERG1 A
currents exhibit inward rectification due to rapid C-type inactivation
(Sanguinetti et al., 1995; Smith et al., 1996; Spector et al., 1996b;
Wang et al., 1997). The state-dependent transitions of HERG1 A have
been modeled to consist of a series of closed states followed by slow
activation to an open state followed by rapid inactivation to a
nonconducting state (Wang et al., 1997). HERG1 A and
Ikr are blocked by methanesulfonanilides, including dofetilide, which has an IC50 in the
low nanomolar range (Jurkiewicz and Sanguinetti, 1993; Spector et al.,
1996a; Wang and Duff, 1996; Ficker et al., 1998; Herzberg et al.,
1998). Previous studies provide evidence that HERG channels are not
blocked by dofetilide when the channel is kept closed and that opening
of the activation gate is necessary for block (Kiehn et al., 1996; Snyders and Chaudhary, 1996).
Dofetilide is effective in the treatment of a range of cardiac
arrhythmias in humans. The recently reported Diamond study (Diamond,
1996) provides evidence that dofetilide treatment may not be associated
with the excess mortality in patients with structural heart disease
that has plagued other antiarrhythmic drugs (Sword trial, 1996;
Preliminary Report of CAST, 1989). An understanding of the
molecular determinants of dofetilide binding to the HERG1 A
K+ channel is relevant to structure-function
analysis and for future design of drugs to block or open the HERG1 A
channel. The purpose of this study was to identify molecular
determinants for high-affinity binding of dofetilide to HERG1 A.
Previous site-directed mutation studies have identified amino acid
residues that alter the affinity of HERG1 A for dofetilide (Ficker et
al., 1998). The S620T mutation, putatively located in the pore helix
near the external mouth of the channel, dramatically decreased the
affinity of the HERG1 A channel for dofetilide (Ficker et al.,
1998). The IC50 of dofetilide for
HERG1 A was 0.32 ± 0.04 µM, whereas it was 248 ± 29 µM
for S620T. Because the S620T mutation also disrupts inactivation, it
was uncertain whether dofetilide interacted directly with this Ser-620
residue or whether the decreased affinity for dofetilide was related
solely to the loss of inactivation (Ficker et al., 1998). All known
mutations of HERG1 A that disrupt inactivation, such as S631A, also
decreased the affinity of the channel for dofetilide (Ficker et al.
1998; Shi et al., 1998). Moreover, all wild-type(WT) EAG-related
channels that do not inactivate also have a low (micromolar) affinity
for dofetilide. In further experiments, Ficker et al. (1998) assessed a
mutation equivalent to HERG S620T in the noninactivating bovine EAG
(BEAG) channel. In those experiments the
IC50 for dofetilide of BEAG T432S was 8 ± 1 µM compared with 32 ± 8 µM for BEAG WT. We hypothesized that
another domain(s) of the channel is the molecular determinants of high
(nanomolar)-affinity binding of dofetilide.
Studies on a range of voltage-gated K+ channels
indicate that amino acids in the S6 domain are molecular determinants
of block by 4-aminopyridine, quinidine, and intracellular application
of tetraethylammonium (TEA) (Choi et al., 1993; Shieh and Kirsch, 1994;
Yeola et al., 1996; Liu et al., 1997; Zhang et al., 1998). Moreover,
amino acids in the S6 of cardiac sodium and L-type calcium channels
also are molecular determinants for binding of local anesthetics and
dihydropyridine calcium channel blockers, respectively (Ragsdale et
al., 1994; Hockerman et al., 1995; McPhee et al., 1995; Peterson et
al., 1996). Accordingly, we hypothesized that a high-affinity
dofetilide binding site was located in the S6 domain of HERG1 A. The
specific objectives of this study were to use site-directed mutagenesis
to define amino acids in the S6 that constitute a molecular determinant
of high-affinity dofetilide binding to HERG1 A, without grossly
altering inactivation properties. In the present study, we identify an
amino acid residue in the S6 of HERG1 A, Phe-656, which is a
determinant of the high-affinity binding of dofetilide.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression in Xenopus Oocytes.
HERG1 A in
pSP64 was obtained from M. T. Keating, University of Utah, Salt
Lake City, UT (Curran et al., 1995). M. T. Site-directed mutagenesis was carried out by overlap extension with the polymerase chain reaction according to Ho et al. (1989). WT and mutated HERG1 A
were transcribed in vitro from the SP6 promoter. The RNA transcripts were injected into Xenopus oocytes and the expressed
currents were analyzed after 1 to 3 days with two-microelectrode
voltage-clamp techniques (Lees-Miller et al., 1997).
Electrophysiologic Recordings.
The oocytes were perfused
with modified frog Ringers solution at room temperature (21-22°C)
containing 114 mmol/l NaCl, 2.5 mmol/l KCl, 1 mmol/l
MgCl2, 1.8 mmol/l CaCl2,
and 10 mmol/l HEPES, pH 7.2, adjusted with NaOH. Niflumic acid was
included (0.15 mM) to block chloride currents. Glass microelectrodes
were filled with 3 M KCl with tip resistances of 0.5 to 2 M
. Oocytes
were clamped with a Geneclamp 500 amplifier and voltage-clamp protocols were generated with pClamp software (Axon Instruments, Foster City,
CA), a Pentium computer, and a Digidata 1200 interface board (Axon
Instruments). Currents were sampled at a rate of 2 KHz. Currents were
filtered with a 4-pole Bessel filter. The oocyte membrane was held at
80 mV between pulses.
80 mV to different depolarizing levels. Each outward current was
elicited by a 2-s depolarizing pulse from potentials of 70 to +30 mV,
in 10-mV steps. To assess kinetics of deactivation, a double-pulse
protocol was used. The first pulse was introduced from a holding
potential of 80 mV to a potential of +50 mV for 2 s followed by
a second pulse to a variety of test potentials ranging from 20 to
120 mV for 2.5 s. The deactivation process was fit to
monoexponential or biexponential functions.
To assess the onset of fast inactivation, expressed channels were
activated and inactivated with a 300-ms pulse to +40. A 25-ms
interpulse to 110 mV was introduced to permit recovery from
inactivation followed by a test pulse to variable potentials ranging
from 70 to +30 mV. This protocol has been previously used to study
inactivation (Smith et al., 1996). The decay of the current was fit to
a mono- or biexponential function. Test pulses were applied every
15 s. To assess recovery from fast inactivation, expressed
channels were activated with a 300-ms pulse to +40 mV followed by a
test pulse to a variable potential ranging from 120 to 20 mV in
10-mV steps. At membrane potentials of less than 80 mV, tail currents
were fit to biexponential functions to account for the rapid increase
in current caused by recovery from channel inactivation and the much
slower decrease in current by deactivation.
After control data were obtained, dofetilide at various concentrations
was introduced. As has been previously reported (Spector et al.,
1996a), the first depolarization yielded little or no reduction in
current when the channel is held constantly at 80 mV during
dofetilide superfusion. Accordingly during dofetilide treatments the
oocytes were pulsed continuously from a holding potential of 80 mV to
+20 mV for 2.5 s with an interpulse interval of 15 s. Because
the development of dofetilide block during superfusion is slow, we
monitored the currents every 5 min and did not obtain records of the
extent of block of the current for 20 min after each change in
dofetilide concentration. We also assessed the effects of potential
"run down" of HERG1 A current during time-dependent evaluations of
these pulse protocols. The extent of run down of HERG1 A during 90 min
of superfusion with Normal Frog Tyrodes was 3.8 ± 1%.
The Kd of binding was fit to the following
model: Icontrol Idofetilide/Icontrol = Bmax *
C/[Kd + C] with
GraphPad Prism Software that involves nonlinear regression modeling
where C indicates the concentration of drug,
Bmax indicates the maximum level of block
of the channel, and Icontrol is the tail current
amplitude at +30 mV; similarly, Idofetilide is
the amplitude of the tail current at +30 mV recorded during dofetilide treatment.
Rationale for Choice of Mutations.
We created four mutations
in the S6 of HERG1 A: M651T, S654L, F656V, and N658V. Table
1 shows a sequence alignment of
homologous regions from the S6 of Shaker B, Kv1.5, Kv2.1, HERG1 A,
BEAG, ELK1, and KcsA. Amino acids that have been reported to alter
affinity for 4-aminopyridine (4-AP), TEA, and quinidine are indicated
in bold (Choi et al., 1993; Shieh and Kirsch, 1994; Yeola et al., 1996;
Liu et al., 1997). Amino acids whose accessibility to
intracellular methanethiosulfonate reagents was altered in the open
versus closed state of the channel are shown in italics and underlined
in Table 1 (Liu et al., 1997).
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). The T505 residue is equivalent
to the T469 site of Shaker (Yeola et al., 1996). In Shaker, the T469S
mutation reduced the affinity for internal TEA by ~2-fold but
substitution with a hydrophobic residue, T469V, produced little effect
(Choi et al., 1993). The M651 residue in HERG1 A is equivalent to T505
in Kv1.5 and accordingly we made the M651T mutation.
The rationale for designing the F656V mutation relates to studies of
Liu et al. (1997) who identified pore sites in Shaker that were only
accessible during channel opening. The V474C mutation, shown in italics
in Table 1, showed the greatest difference in modification rate between
the open and closed states of the channel. Thus, access to this amino
acid was strikingly dependent on gating. Moreover, the V474C Shaker
current was blocked by Cd2+ but only when the
channels were opened. Similarly, Cd2+ could only
be released from the channel when it was opened. A similar phenomena of
block and release of dofetilide from IKr and
HERG1 A occurs when the channel is opened (Carmeliet, 1992; Spector et
al., 1996a). The Val-474 residue of Shaker is equivalent to
Phe-656 of HERG1 A. Moreover Shieh and Kirsch (1994) reported that the
I405V mutation of Kv2.1 increased 4-AP affinity, independent of changes
in gating. From these data, the authors concluded that this site and
another site in the S5 would be amino acid residues critical for 4-AP
binding. The Ile-405 residue of Kv2.1 is equivalent to Phe-656 of HERG1
A. Accordingly, we created the F656V mutation.
The structural determinants of quinidine affinity for the sodium
channel also have been examined. Residues Ile-764 and Ile-771 of the IV
S6 are important determinants of molecular binding of quinidine
to the sodium channel (Ragsdale et al., 1994; McPhee et al.,
1995). According to the proposed alignment of this sodium channel with
Kv1.5, these residues are equivalent to Gly-648 and Ser-654 of HERG1 A
(Yeola et al., 1996). Moreover, Shieh and Kirsch (1994) reported that
the mutation L403M of Kv2.1, a site equivalent to Ser-654 of HERG1
A, changed the affinity of Kv2.1 for internal TEA. Accordingly, we
also created the S654L mutation of HERG1 A.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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F656V Alters Affinity of HERG1 A for Dofetilide and Quinidine but
not External TEA.
A number of compounds block HERG1 A and
IKr, including dofetilide; external TEA
(TEAe), which interacts with the inactivation gate at Ser-631; and other nonspecific K+ channel
blockers, such as quinidine. Figure 1
shows the extent of block of WT and the F656V mutation by dofetilide
(A), quinidine (B), and TEAe (C). The F656V mutation
increased the IC50 for dofetilide by two orders
of magnitude from 0.125 ± 0.003 µM for WT to 15 ± 3 µM.
Similarly, the IC50 for quinidine was increased
from 8 ± 4 for WT to 219 ± 65 µM for the F656V mutation.
In contrast, the IC50 for
TEAe was similar for WT (51 ± 10 mM) and
F656V (36 ±10 mM, NS). The dose-response relationships for
TEAe block did not fit a first-order binding
isotherm because of the low affinity of HERG1 A for
TEAe. Similar dose-response relationships of
TEAe for HERG1 A have been previously reported by
Smith et al. (1996). Because saturation was not observed, the
measurement of affinity of HERG1 A and F656V for
TEAe are considered estimates. However, a review
of the raw data indicates that the extent of block by TEAe for WT and F656V shows little, if any
difference. These data indicate that F656V is a molecular determinant
for high-affinity dofetilide binding. Moreover, the F656V mutation
alters affinity of the channel for dofetilide and quinidine but not for
TEAe. In contrast, previous studies have reported
that mutation of Ser-631 alters affinity of the channel for dofetilide
and TEAe (Smith et al., 1996; Ficker et al.,
1998) but we find that S631A does not alter the affinity for quinidine
(see below). Because the F656V mutation did not change response to
TEAe, it seemed likely that inactivation was
intact. Accordingly, we assessed the inactivation and other gating
characteristics of the channel.
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F656V Is Inwardly Rectified with Inactivation Characteristics
Similar to HERG1 A.
If F656V inactivates in a manner similar to
HERG1 A, we expect that the time-dependent currents would be inwardly
rectified. Figure 2 shows typical
currents of WT in the left column and F656V in the right column. From a
holding potential of 80 mV, currents were evoked by a series
depolarizing potentials, P1, for 2500 ms to potentials between 70 and
+50 mV followed by the P2 pulse to a holding potential of 60 mV. The
current measured at the end of P1 was related to the depolarizing
potentials. These mean current-voltage data are shown in Fig. 2, E and
F. Inward rectification was evident for both WT and F656V. From these
data, we concluded that the characteristic of inward rectification is
common to WT and F656V.
|
and Spector et al.
(1996b). No substantial difference in the time course of onset of
inactivation was noted. Data were best fit to a monoexponential
function. The mean time constants for the onset of inactivation are
plotted against voltage in Fig. 3C and are similar for WT and F656V.
For example, the mean time constants at 20 mV were 13 ± 1 ms
and 11 ± 1 ms for WT and F656V, respectively, and at +20 mV were 9 ± 1 ms for WT and 8 ± 0.2 ms for F656V. These data indicate that the characteristics of onset of inactivation are similar in WT and
F656V.
|
was used. Representative examples of the time course of recovery from
inactivation and its fitting to a monoexponetial process are shown in
Fig. 4. The general character of recovery from inactivation was similar in WT and F656V. The mean time constants for recovery from inactivation are plotted against voltage for WT and
F656V in Fig. 4C. The time constants of recovery from inactivation were
significantly slower for WT than for F656V. For example, the mean time
constant at 20 mV was 13 ± 1 ms for WT and 8 ± 0.3 ms for F656V
(P < .01, Student's t test). These data
indicate that F656V alters the time course of recovery from
inactivation.
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Activation Characteristics of F656V Compared with WT.
To
assess whether the F656V mutation had altered the process of
activation, the peak of the tail currents at the onset of the P2 pulse
was related to the P1 potentials and presented for WT in Fig. 2E and
F656V in Fig. 2F. The voltage-dependence of the activated tail currents
of WT were well fit to a Boltzman function with V1/2
for activation of 26 ± 7 mV (slope 9 ± 6), whereas the
V1/2 for F656V was 9 ± 6 mV (slope 15 ± 1 mV). These data indicate that the F656V mutation significantly shifts
the voltage dependence of activation to more depolarized potentials.
Deactivation.
To assess whether the F656V mutation had altered
the process of deactivation, the time constants of the decay of the
tail currents at the onset of the P2 pulse were assessed.
Representative examples of the deactivation currents are shown for WT
in Fig. 2C and for F656V in Fig. 2D. At 80 mV, the 
value of the
fast component of deactivation for WT (173 ± 9 ms) was slower
than for F656V (75± 5 ms), whereas at 40 mV the values were
1088 ± 240 and 316 ± 9 ms, respectively (P < .01).
Other S6 Mutations Tested Have Little or Modest Effects on Affinity for Dofetilide. A number of other S6 mutations were studied, including M651T, S654L, and N658V. The IC50 of these mutant channels for dofetilide were 0.120 ± 0.08, 0.3 ± 0.1, and 0.119 ± 0.7 µM, respectively.
Figure 5 shows the character of activating currents of these mutations. The deactivation time constant of M651T is markedly prolonged, whereas in the N658V mutation it is abbreviated. The mean values of the rapid component of deactivation
at 90 mV for WT, M651T, and N658V were 116 ± 7, 508 ± 205, and 16 ± 6 ms, respectively. Thus, the deactivation can
be altered by 50-fold (from 16 to 508 ms) and yet the
IC50 for dofetilide of M651T and N658V are the
same as WT. The V1/2 for activation as measured from the tail currents were shifted to 32 ± 6 mV for M651T, to 18 ± 2 mV for N658V, and to 6 ± 5 mV for S654L compared
with 26 ± 7 mV for WT, but their IC50
values were similar. Thus, shortening or prolonging deactivation or
shifting the voltage dependence of activation did not correlate with a
change in the affinity of the channel for dofetilide.
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20 mV the values of onset of inactivation are identical even
though the IC50 for dofetilide differs by two orders of magnitude (0.120 ± 0.08 µM/l for M651T and 15 µM
for F656V). Moreover, shifts in voltage dependence of inactivation also
do not appear to correlate with affinity for dofetilide. For example,
the S654L mutation has a shifted voltage dependence of onset of
inactivation and a shifted IC50 for dofetilide;
however, the N658V mutation has a very similar shift in the onset of
inactivation but the IC50 for dofetilide is
similar to that of WT, 0.119 ± 0.7 µM. Thus, for these
mutations, there is no correlation between alteration in the character
of onset of inactivation and the IC50 for dofetilide.
Figure 4C compares the kinetics of recovery from inactivation of mutant
and WT HERG1 A channels. The M651T mutation has a voltage-dependent
recovery from inactivation, which is similar to F656V over voltages
from 90 to 50 mV. At 70 mV, the of recovery from inactivation
is 6 ± 0.2 for F656V and 6 ± 0.5 ms for M651T, even though
the IC50 for F656V is two orders of magnitude different than M651T. In addition shifts of the voltage dependence of
recovery from inactivation to the right, as seen with N658V do not
correlate with a change in the IC50 for
dofetilide. Thus, there is no correlation between alteration in the
character of recovery from inactivation and the
IC50 for dofetilide.
Pharmacologic Responses to S631A and to Double Mutant
F656V/S631A.
To assess the effects of disrupting inactivation, the
S631A mutation was made. The IC50 values of
TEAe, a drug known to interact with the S631 site
(Smith et al., 1996); dofetilide, a drug also known to have its
IC50 altered by S631A (Ficker et al., 1998); and
quinidine, a nonspecific potassium channel blocker were assessed in
HERG1 A and in the S631A and F656V mutants. In this study, the S631A
mutation decreased the affinity for dofetilide to 21 ± 4 µM, a
result similar to that previously reported (Ficker et al.,
1998). However, the IC50 for quinidine is
not affected by the S631A mutation (8 ± 2 µM for WT and 10 ± 1 µM for S631A). In contrast to the S631A mutation, the F656V
mutation, as reported above, decreases the affinity for quinidine and
dofetilide but not for TEAe. Moreover, affinity
for TEAe is altered by S631A, affinity for
quinidine is altered by F656V, and affinity for dofetilide is altered
by mutations in either sites. These data indicate that these different
drugs have different responses to the S631A and the F656V mutations.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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New Information Provided by This Study.
Previous studies have
identified mutations such as S631A and S620T that alter the affinity of
HERG1 A for dofetilide, but also disrupt inactivation (Ficker
et al., 1998; Zou et al., 1998). The decreased affinity of the channel
for dofetilide produced by the F656V mutation was not associated with
disrupted inactivation. The F656V mutation alters affinity of the
channel for dofetilide and quinidine but not
TEAe, whereas the S631A mutation alters affinity
of the channel for dofetilide and TEAe (Smith et
al., 1996; Ficker et al., 1998) but not for quinidine. To address
whether the F656V mutation alters the IC50 for
dofetilide in a channel that does not inactivate (S631A), the double
mutation S631A/F656V was made, and the IC50 for
dofetilide of the double mutant was not grossly different than is
observed for the individual mutations. These data support the notion
that allosteric changes occurring during the process of inactivation
appear to be necessary for high-affinity dofetilide binding.
F656V Alters Affinity for Dofetilide Unrelated to Changes in
Channel Gating.
Previous studies have demonstrated that mutation
of S620 to T dramatically decreases HERG1 A's affinity for dofetilide
(Ficker et al., 1998). However, the S620T mutation also
disrupts inactivation (Ficker et al., 1998), therefore, it was
uncertain whether dofetilide interacted directly with this S620 residue
or whether the decreased affinity for dofetilide was related solely to
the loss of inactivation. In contrast, the F656V mutation decreases the
affinity of HERG1 A without a disruption of the inactivation
characteristics. Moreover, it is unlikely that quantitative differences
in activation, deactivation or onset and recovery from inactivation are
responsible for the reduced affinity of F656V for dofetilide because
mutations of nearby amino acids that either prolong or shorten these
gating kinetics have no effect on the IC50 of dofetilide.
Phe-656 Is Necessary but not Sufficient for High-Affinity
Dofetilide Binding: Contribution of Inactivation.
The structure of
the KcsA K+ channel has been determined by X-ray
crystallography (Doyle et al., 1998). The apparent structure of IRK1,
as determined by mutagenesis, differs significantly from KcsA (Minor et
al., 1999). However, KcsA is more similar in its primary structure to
voltage-gated channels than to IRK1, suggesting that the topologic
features of KcsA may provide a structural framework for considering the
molecular determinants of interaction of dofetilide with HERG Phe-656.
Thr-107 in KcsA aligns with Phe-656 of HERG1 A. Thr-107 is located near
the intracellular pore mouth and faces the pore stream and is at the
apex of the "inverted teepee tent" configuration of the KcsA
potassium channel, placing it at or near the narrowest portion of the
intracellular mouth of the channel. Therefore, binding of a large
organic compound at this site might be expected to occlude the channel.
The presence of a phenylalanine at this site in HERG1 A appears to be
important in creating a binding site for dofetilide and quinidine.
Dofetilide consists of a long aliphatic chain with substituted benzene
rings at each end. Benzene rings are known to stack via pi bonds and it
is possible that the benzene head groups of dofetilide could interact
with the benzene ring of phenylalanine. Interestingly, none of the Shaker, Shab, Shaw, Shal family of K+ channels
has a phenylalanine at a position equivalent to Phe-656. This may
account for the relative specificity of dofetilide for HERG1 A. However, the noninactivating BEAG and ELK1 channels, which also have an
F in positions equivalent to Phe-656 (Table 1), are relatively
insensitive to methanesulphonanilides. Therefore, the presence of
Phe-656 is necessary for high-affinity dofetilide binding, but other
characteristics of the channel also must contribute to differences in
dofetilide binding. Because ELK1 and BEAG also do not inactivate, these
data suggest that there are two characteristics that are determinants
of high-affinity dofetilide binding, the Phe-656 amino acid and
allosteric changes that take place during the process of inactivation.
;
Kiehn et al., 1996). Ficker et al. (1998) have previously considered
whether inactivation could contribute to dofetilide block or whether
all of the characteristics of dofetilide block can be explained by
open-channel block. Evidence against open-channel block was that
dofetilide did not produce significant changes in open-channel time
(Kiehn et al., 1996). To account for these observations, Ficker et al.
(1998), proposed that dofetilide binds to a preinactivated state of the
channel, which is consistent with the single channel-bursting behavior
of the channel. The presence of a preinactivated state as a determinant
of binding of dofetilide is consistent with our findings.
Proposed Model of Dofetilide Binding to HERG1 A.
Although a
preinactivated state of the channel may be a determinant of dofetilide
binding, we present an alternative model that proposes that sequential
allosteric changes, including activation and subsequent inactivation
(or preinactivation) are necessary for high-affinity dofetilide
binding. In this model, opening of the activation gate allows access of
dofetilide to the inner vestibule of the channel. Thereafter,
allosteric changes associated with inactivation allow association of
dofetilide with its high-affinity binding site. Following
repolarization, the channels reactivate and close allowing dissociation
of dofetilide from the high-affinity site, but the activation gate must
reopen to allow dofetilide to leave the inner vestibule of the channel
pore. In this model, the activation gate determines access of
dofetilide to the inner vestibule of HERG1 A and as such is a critical
determinant of association with and dissociation from the pore, whereas
the process of inactivation appears to be necessary for association of
dofetilide with its high-affinity binding site in the S6. Consistent
with this model are the observations that an opening of the activation gate is necessary for dofetilide block and unblock (Kiehn et al., 1996;
Snyders and Chaudhary, 1996). The evidence that allosteric changes
associated with the inactivation process are necessary for block is
that all known mutations of HERG1 A that disrupt inactivation also
decrease the affinity of the channel for dofetilide independent of
whether or not they face the pore stream. Moreover, all WT EAG-related
channels that do not inactivate but have an Phe at positions equivalent
to Phe-656 also have a low (micromolar) affinity for dofetilide,
whereas the inactivating channels ERG2 and ERG3 are sensitive to
nanomolar concentrations of methanesulfonanilides (Shi et al., 1997,
1998). Our model does not define whether the inactivation gate directly
alters access to the receptor at Phe-656 or whether allosteric changes
in the entire pore that occur during inactivation alter access of
dofetilide to its high-affinity receptor. An alternative explanation of
how inactivation could alter dofetilide binding to the S6 is that
inactivation stops potassium flux across the pore. Ongoing potassium
flux could inhibit binding. In summary, in this model conformational
changes in the activation gate determine access of dofetilide to the
inner vestibule, whereas a transition state involved in inactivation
appears necessary for binding of dofetilide to the high-affinity site
in the S6.
; Snyders et al., 1996). Our model requires two
allosteric steps to achieve dofetilide block; first the channel must
open to allow access of dofetilide to the inner vestibule, and then the
channel must transition to an inactivated state. At +80 mV, the
current-voltage relationship of HERG indicates that virtually all
channels will be in the inactivated state (Sanguinetti et al., 1995),
with only rare openings (a state necessary for dofetilide binding). In
contrast, at +10-mV channels are in equilibrium between open and
inactivated states and so dofetilide can block the channel. In
conclusion, the Phe-656 residue of HERG1 A is a molecular determinant
of high-affinity dofetilide binding.
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Acknowledgments |
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We are appreciative of the helpful suggestions of Drs R. S. Clark, A. M. Gillis, and R. S. Sheldon and the technical assistance of Lilong Tang and Kevin Schade.
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Footnotes |
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Received July 28, 1999; Accepted November 9, 1999
This work was supported by Medical Research Council of Canada, the Heart and Stroke Foundation of Alberta, and the Andrews Family Professorship in Cardiovascular Medicine.
Send reprint requests to: H. J. Duff, M.D., F.R.C.P.(C), Department of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1. E-mail: hduff{at}ucalgary.ca
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Abbreviations |
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EAG, ether a-go-go; WT, wild type; TEA, tetraethylammonium; TEAe, external TEA; 4-AP, 4-aminopyridine; TEAe, tetraethylammonium external.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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subunit in fast inactivation.
J Biol Chem
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) and the tail currents (
) for
HERG1 A in E and F656V in F. Both the time-dependent currents and the
tail currents show inward rectification.
), F656V (
),
S654L (
), N658V (
). There was no significant
difference in the onset of inactivation comparing HERG1 A and F656V.
The voltage-
