Action potential clamp and mefloquine sensitivity of recombinant 'I KS' channels incorporating the V307L KCNQ1 mutation.

Abstract

The slow delayed rectifier potassium current, 'I(Ks)', contributes to repolarisation of cardiac ventricular action potentials and thereby to the duration of the QT interval of the electrocardiogram. Mutations to I(Ks) channel subunits occur in clinically significant cardiac repolarisation disorders. The short QT syndrome (SQTS) is associated with accelerated ventricular repolarisation and with an increased risk of arrhythmia and sudden death. The SQT2 variant of the SQTS has been linked to a gain-of-function amino-acid substitution (V307L) in the KCNQ1-encoded I(Ks) channel alpha-subunit. This study reports the first action potential (AP) voltage-clamp comparison between wild-type (WT) and V307L KCNQ1 (co-expressed with KCNE1 to recapitulate I(Ks)) and identifies an effective pharmacological inhibitor of recombinant 'I(Ks)' channels incorporating the V307L KCNQ1 mutation. Perforated-patch voltage-clamp recordings at 37 degrees C of whole-cell current carried by co-expressed KCNQ1 and KCNE1 showed a marked (-36 mV) shift in half-maximal activation for V307L compared to WT KCNQ1; a significant slowing of current deactivation was also observed. Under AP clamp, peak repolarising current was significantly augmented for V307L KCNQ1 compared to WT KCNQ1 for both ventricular and atrial AP commands, consistent with an ability of the V307L mutation to increase repolarising I(Ks) in both regions. The quinoline agent mefloquine inhibited WT KCNQ1+KCNE1 with an IC(50) of 3.4 muM compared to 3.3 muM for V307L KCNQ1+KCNE1 (P >0.05). This establishes mefloquine as an effective inhibitor of recombinant 'I(Ks)' channels incorporating this SQT2 KCNQ1 mutation.

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INTRODUCTION
A number of different potassium channels contribute to
repolarisation of cardiac ventricular action potentials (1). The
rapid and slow delayed rectifier K+currents (denoted 'IKr' and 'IKs'
respectively) play important roles in determining repolarisation
over the ventricular action potential (AP) plateau phase and,
thereby, in determining the QT interval of the electrocardiogram
(ECG) (1, 2); subsequent terminal AP repolarisation is mediated
predominantly by the inwardly rectifying K+current, IK1(1). IKs
plays an important role in autonomic regulation of ventricular
repolarisation (3, 4); it also provides a ventricular 'repolarisation
reserve' that restricts excessive action potential prolongation
under conditions of reduced IKr(5, 6). Functional IKschannels are
comprised of an α subunit encoded by KCNQ1 and a β subunit
encoded by KCNE1 (7, 8) with the gene products also
respectively denoted KCNQ1 and KCNE1). Mutations in either
gene can lead to clinically significant cardiac repolarisation
disorders (1, 2, 9, 10).
The short QT syndrome (SQTS) is a cardiac repolarisation
disorder in which patients exhibit shortened QT intervals on the
electrocardiogram and an increased incidence of cardiac
arrhythmias and sudden death in the absence of structural heart
disease (10, 11). Gain-of-function mutations to three K+channel
genes (KCNH2, KCNQ1 and KCNJ2) have been identified in
SQTS patients, giving rise to the SQT1, SQT2 and SQT3 variants
of the syndrome respectively (12-16). The adult SQT2 variant was
identified in a 70 year old male who was resuscitated from an
episode of ventricular fibrillation (15). No physical abnormalities
were present and in sinus rhythm normal conduction intervals
were observed (15). However, marked abbreviation of ventricular
repolarisation was observed both on initial presentation (with a
rate corrected QT (QTC) interval of 302 ms) and over a three year
follow-up period (15). Genetic testing identified a G?C
substitution at nucleotide 919 of the KCNQ1 gene, giving rise to a
single amino-acid substitution (valine?leucine) at position 307
(V307L) of the KCNQ1 protein (15).
Residue V307 of KCNQ1 is located in the channel's pore
helix and the V307L mutation was reported to alter activation
time-course and voltage-dependence, giving rise to increased
current through co-expressed V307L-KCNQ1+KCNE1 channels
in conventional voltage-clamp experiments (15). Although
computer simulations have predicted accelerated ventricular
repolarisation and shortened refractory periods at cell and tissue
JOURNAL OF PHYSIOLOGYAND PHARMACOLOGY 2010, 61, 2, 123-131
www.jpp.krakow.pl
A. EL HARCHI1, M.J. MCPATE1,2, Y.H. ZHANG1, H. ZHANG3, J.C. HANCOX1
ACTION POTENTIAL CLAMPAND MEFLOQUINE SENSITIVITY OF RECOMBINANT
'IKS' CHANNELS INCORPORATING THE V307L KCNQ1 MUTATION
1Department of Physiology and Pharmacology and Cardiovascular Research Laboratories, School of Medical Sciences, University
Walk, University of Bristol, UK; 2Novartis Institutes for Biomedical Research, Horsham, West Sussex, UK; 3Biological Physics
Group, School of Physics and Astronomy, The University of Manchester, Manchester, UK
The slow delayed rectifier potassium current, 'IKs', contributes to repolarisation of cardiac ventricular action potentials
and thereby to the duration of the QT interval of the electrocardiogram. Mutations to IKschannel subunits occur in
clinically significant cardiac repolarisation disorders. The short QT syndrome (SQTS) is associated with accelerated
ventricular repolarisation and with an increased risk of arrhythmia and sudden death. The SQT2 variant of the SQTS has
been linked to a gain-of-function amino-acid substitution (V307L) in the KCNQ1-encoded IKschannel α-subunit. This
study reports the first action potential (AP) voltage-clamp comparison between wild-type (WT) and V307LKCNQ1 (co-
expressed with KCNE1 to recapitulate IKs) and identifies an effective pharmacological inhibitor of recombinant 'IKs'
channels incorporating the V307L KCNQ1 mutation. Perforated-patch voltage-clamp recordings at 37°C of whole-cell
current carried by co-expressed KCNQ1 and KCNE1 showed a marked (-36 mV) shift in half-maximal activation for
V307L compared to WT KCNQ1; a significant slowing of current deactivation was also observed. Under AP clamp,
peak repolarising current was significantly augmented for V307LKCNQ1 compared to WT KCNQ1 for both ventricular
and atrial AP commands, consistent with an ability of the V307L mutation to increase repolarising IKsin both regions.
The quinoline agent mefloquine inhibited WT KCNQ1+KCNE1 with an IC50of 3.4 µM compared to 3.3 µM for V307L
KCNQ1+KCNE1 (P >0.05). This establishes mefloquine as an effective inhibitor of recombinant 'IKs' channels
incorporating this SQT2 KCNQ1 mutation.
Key words: arrhythmia, antiarrhythmic, KCNQ1, KCNE1, KvLQT1, mefloquine, minK, QT interval, short QT syndrome,
sudden death

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levels as a result of the V307LKCNQ1 mutation (15, 17), to-date
there has been no direct characterisation of increased repolarising
current during physiological waveforms with this mutation. By
contrast, for both the SQT1 KCNH2 and SQT3 KCNJ2 mutations
the action potential (AP) voltage-clamp technique has been used
to provide direct insight into altered timing and magnitude of
repolarising currents during AP waveforms (12, 18-21). In
addition, whilst in vitro investigations have identified effective
pharmacological inhibitors of SQT1 and SQT3 mutant K+
channels (21-24), no such pharmacological data have yet been
reported for channels incorporating SQT2 V307L mutant
KCNQ1 subunits. The present study was undertaken to address
these issues. Thus we report here, for the first time: (i) AP clamp
data for V307L-KCNQ1-KCNE1 channels and (ii) an effective
pharmacological inhibitor of these channels.
MATERIALS AND METHODS
Maintenance of cells expressing wild-type (WT) and V307L
KCNQ1 together with KCNE1
WT KCNQ1 (in pIRES expression vector) was kindly
provided by Dr J Bahranin. Plasmid encoding the common S38
variant of KCNE1 (in pCR3.1) (25;26) was kindly donated by Dr
F Toyoda. The V307Lmutation was introduced into KCNQ1 using
QuikChange®(Stratagene; mutagenesis primer of: 5'GCT GTG
GTG GGG GCT GGT CAC AGT CAC 3'). DNA was sequenced
for the full length of the KCNQ1 insert to ensure that only the
correct mutation had been made (Eurofins MWG Operon).
Chinese Hamster Ovary (CHO) cells were passaged using a
non-enzymatic agent (Enzyme Free, Chemicon®International)
and then maintained as described previously. Twenty-four hours
after plating cells out, the cells were transiently co-transfected
2:1 with KCNE1 and either WT or V307L-KCNQ1 (0.7 µg of
each KCNQ1 construct was used) using LipofectamineTMLTX
(Invitrogen) according to the manufacturer's instructions.
Expression plasmid encoding CD8 was also added (in pIRES,
donated by Dr I Baro and Dr J Barhanin) to be used as a
successful marker of transfection. Cells were plated onto small
sterilised collagen-coated glass coverslips 6 hours after
transfection and recordings were made after at least 24 hours
incubation at 37°C. Successfully transfected cells (positive to
CD8) were identified using Dynabeads®(Invitrogen).
Electrophysiological recordings
For perforated patch-clamp recording cells were continuously
superfused (at 37°C) with an external solution containing (in
mM): 140 NaCl, 4 KCl, 2.5 CaCl2, 1 MgCl2, 10 Glucose and 5
124
Fig. 1. WT and V307L IKCNQ1-KCNE1under conventional voltage clamp. (A) Upper traces show records of WT (Ai) and V307L IKCNQ1-KCNE1
(Aii) elicited by 3-s duration voltage clamp commands applied from -80 mV to a range of potentials (lower traces). The numbers adjacent
to each of the currents shown in Ai and Aii indicate the command voltage of the corresponding 3-s command used to elicit IKCNQ1-KCNE1. A
brief 50 ms pulse to -40 mV from the holding potential of -80 mV was incorporated prior to each test command in order to monitor
instantaneous leak current, which can be seen to be negligible in these recordings. Following each test command a 5-s repolarising step to
-40 mV was incorporated, enabling observation of deactivating tail currents. Successive steps in the protocol were applied every 10 s. (B)
End pulse current-voltage (I-V) relations for WT and V307L IKCNQ1-KCNE1(n=13 and 9 and denoted by 'squares' and 'triangles' respectively.
*** denotes P<0.001, ** denotes P<0.01, * denotes P<0.05). The protocol used to determine the I-V relations is similar to that shown in
'A', with successive 10 mV steps from -70 mV to + 60 mV (test pulse frequency of one pulse every 10 s). (C) Normalized current-voltage
(I-V) relations for peak 'tail' amplitudes for WT and V307L IKCNQ1-KCNE1(n=13 and 9 and denoted by 'squares' and 'triangles' respectively,
voltage protocol as in B). Normalization was carried out as follows: for each cell the amplitude of IKCNQ1-KCNE1on repolarisation to -40 mV
following different test potentials was normalized to the maximum current observed during application of the protocol. The resulting data
were fitted with equation 1 to give the V0.5and k values in the 'Results and Discussion' text.

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HEPES (titrated to pH 7.45 with NaOH) (21). Patch-pipettes
(Corning 7052 glass, AM Systems) were pulled and heat-polished
(Narishige MF83) to 2.5-4 MΩ; pipette dialysate contained (in
mM): 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (titrated
to pH 7.2 using KOH) (21) and 225 µg/mL amphotericin B.
Recordings of KCNQ1+KCNE1 current (IKCNQ1-KCNE1) were made
using an Axopatch 200 amplifier (Axon Instruments) and a
CV201 head-stage. Between 70-80% of pipette series resistance
was compensated. Voltage-clamp commands were generated
using 'WinWCP' (John Dempster, Strathclyde University). Human
ventricular and atrial APwaveforms used as voltage commands in
AP clamp experiments were generated using established
ventricular (27) and atrial (28) cell mathematical models. IKCNQ1-
KCNE1 elicited under AP clamp was obtained using 'p/4' leak
subtraction (cf (29)).
Experimental compounds
Mefloquine-HCl powder (Sigma) was dissolved in DMSO to
produce an initial stock solution of 50 mM which was diluted to
produce stock solutions ranging down to 100 µM. The
mefloquine-HCl stock solutions were diluted at least 1:1000-
fold with Tyrode's solution to achieve concentrations stated in
the Results. External solutions were applied using a home-built,
warmed and rapid solution exchange device. Control solution for
experiments with mefloquine contained DMSO (in order to
constitute a vehicle control) at a concentration matching that in
the mefloquine-containing solutions tested.
Data analysis
Unless otherwise stated in the text data are presented as
mean±standard error of the mean (S.E.M.). The voltage
dependence of IKCNQ1-KCNE1activation was determined by fitting
the values of IKCNQ1-KCNE1tail currents (normalised to peak IKCNQ1-
KCNE1tail value and plotted against voltage) with a Boltzmann
equation of the form:
[1]I = IMAX/(1+exp (V0.5-Vm)/k)
where I is the IKCNQ1-KCNE1 tail amplitude following test
potential Vm, IMAXis the maximal IKCNQ1-KCNE1observed, V0.5is
the potential at which IKCNQ1-KCNE1was half-maximally activated,
and k is the slope factor for the relationship. The time-course of
IKCNQ1-KCNE1activation and deactivation were fitted with standard
bi-exponential equations. Concentration-response relations were
fitted with a standard Hill equation to obtain half-maximal
inhibitory concentration (IC50) and Hill-coefficient (nH) values.
Statistical analysis was performed using analysis of variance or
t-tests as appropriate (Graphpad Prism v5). P values of less than
0.05 were taken as statistically significant.
RESULTS
Effects of V307L on IKCNQ1-KCNE1under conventional voltage clamp
The functional consequences of the V307L mutation for
IKCNQ1-KCNE1were first characterised using conventional whole-
cell voltage clamp protocols. Fig. 1A shows representative
traces for WT (Fig. 1Ai) and V307L (Fig. 1Aii) IKCNQ1-KCNE1
elicited by depolarisation from -80 mV to the test potentials
shown. As shown in Fig. 1Ai, a test pulse to -30 mV elicited
relatively little WT IKCNQ1-KCNE1whilst, in contrast, markedly
larger IKCNQ1-KCNE1 was evident for V307L KCNQ1 at this
voltage. Mutant IKCNQ1-KCNE1was also markedly larger than WT
current at 0 mV. Fig. 1B shows mean end-pulse current-voltage
(I-V) relations for WT and V307L IKCNQ1-KCNE1, showing that
125
Fig. 2. Voltage dependence of WT and
V307L IKCNQ1-KCNE1 deactivation. (A)
Plots against voltage of fast (Ai) and slow
(Aii) time constants of activation for WT
and V307L IKCNQ1-KCNE1(n=13 and 9 and
denoted by 'squares' and 'triangles'
respectively, voltage protocol as in B).
Time constant ("tau") values were
obtained from standard bi-exponential
fitting of the outward currents that
developed on membrane depolarisation
(* denotes P<0.05 versus WT). (B) Plots
against voltage of time constants of
deactivation for WT and V307L IKCNQ1-
KCNE1 (n=13 and 9 and denoted by
'squares' and 'triangles' respectively,
voltage protocol shown as an inset in Bi.
This was comprised of a 2 second
depolarisation to +40 mV to activate
IKCNQ1-KCNE1, followed by a 4 second
duration step to a series of different
voltages, at each of which the rate of
current deactivation was quantified). Tail
current decline (deactivation) was fitted
with a bi-exponential equation to derive
values for fast (Bi) and slow (Bii)
components of deactivation that were
then plotted against the corresponding
voltage (***denotes P<0.001, ** P<0.01;
* P<0.05 between WT and V307L).

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across a range of potentials between -10 and +60 mV V307L
IKCNQ1-KCNE1 was significantly greater than the corresponding
WT current. Fig. 1C shows steady-state voltage-dependent
activation plots for WT and V307L IKCNQ1-KCNE1, with fits to
normalized 'tail' current data with equation 1 to derive V0.5and
k values. There was a marked left-ward shift in activation V0.5
for V307L IKCNQ1-KCNE1 (-23.46±0.73 mV; n=9; P<0.001)
compared to WT IKCNQ1-KCNE1(V0.5of +12.52±0.41 mV; n=13),
without any significant change to the slope of the relationship
(with k values for V307L and WT IKCNQ1-KCNE1respectively of
16.49±0.67 mV and 13.69±0.37 mV; P>0.1). This left-ward
shift in the voltage-dependence of activation can account for
the greater currents at negative voltages for V307L shown in
Fig. 1A.
The effects of the V307L mutation on the time-course of
IKCNQ1-KCNE1activation and deactivation were assessed using bi-
exponential curve fitting of the time-course of current
development on depolarisation (activation) and decline on
repolarisation (deactivation). Similar to Bellocq and colleagues
(15), we observed a tendency for both fast and slow activation
time-constants to be smaller in magnitude for V307L than for
WT IKCNQ1-KCNE1, particularly at negative membrane voltages
(Fig. 2Ai, Aii). However, the time-course of activation for WT
IKCNQ1-KCNE1exhibited considerable heterogeneity in our study, so
at the majority of test potentials examined the differences
between WT and V307L IKCNQ1-KCNE1activation time-constants
did not attain statistical significance. By contrast, however,
deactivation of V307L IKCNQ1-KCNE1was markedly slower than
that of WT IKCNQ1-KCNE1. This is evident both in the current
records shown in Fig. 1A and in the time-constant plots shown
in Fig. 2Bi and Bii. The fast time-constant of deactivation was
slower for V307L than for WT IKCNQ1-KCNE1across all plotted
voltages (-60 mV to 0 mV), whilst the slow time constant was
also larger for V307L at four of seven test voltages at which
deactivation time-course was examined.
AP clamp of WT and V307L IKCNQ1-KCNE1
The results of AP clamp experiments using an epicardial
ventricular AP waveform (19, 21) are shown in Fig. 3. Fig. 3Ai
and 3Aii show WT and V307L IKCNQ1-KCNE1each overlain on the
ventricular AP command. Very little outward current occurred
immediately on depolarisation, but current then increased
progressively throughout the AP plateau reaching peak
amplitude late in the plateau phase. V307L IKCNQ1-KCNE1showed a
similar profile to that of WT IKCNQ1-KCNE1, except that the current
126
Fig. 3. WT and V307L IKCNQ1-KCNE1 during
ventricular AP clamp. (A) Profile of WT (Ai)
and V307L(Aii) IKCNQ1-KCNE1(the thicker of the
pair of overlaid traces) during an applied
ventricular AP command (the thinner of the
pair of overlaid traces; APcommand frequency
of 1Hz). Each of (Ai) and (Aii) has its own
time axis, whilst the current axis (shown to the
left of (Ai)) and voltage axis (shown to the
right of (Aii)) apply to both panels. (B) Plots of
the corresponding instantaneous current-
voltage (I-V) relations for WT (Bi) and V307L
(Bii) IKCNQ1-KCNE1 during ventricular AP
repolarisation phase. Instantaneous I-V
relations show continuous plots of current
recorded during AP repolarisation and the
direction of repolarisation on each plot is
indicated by the filled 'arrow' symbols. (C)
Comparison of mean (±S.E.M) maximal
repolarising current during AP repolarisation
between WT and V307L IKCNQ1-KCNE1 (n=13
and n=12 respectively; P<0.01).

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amplitude was markedly larger. Fig. 3Bi and 3Bii show
instantaneous I-V plots of current during AP repolarisation for
the same cells shown in Figs. 3Ai and 3Aii. For both WT and
V307L IKCNQ1-KCNE1maximal currents occurred between ~ 0 and
+10 mV; WT maximal repolarising current occurred at +5.9±0.9
mV (n=13), whilst V307L maximal repolarising current
occurred at +4.8±1.0 mV (n=12; P>0.1 vs. WT). Fig. 3C shows
mean plots of the maximal amplitudes of WT and V307L IKCNQ1-
KCNE1, demonstrating a significantly greater (>2-fold) maximal
repolarising current with the SQT2 mutation.
Although atrial arrhythmia was not reported for the patient
in whom the V307L mutation was first identified (15), some
SQTS patients do experience atrial fibrillation (10, 11).
Consequently, and also for purposes of comparison with
ventricular AP clamp data, we studied the effects of the V307L
KCNQ1 mutation on the profile of IKCNQ1-KCNE1during an atrial
AP command waveform. The results of atrial AP clamp
experiments are shown in Fig. 4. For six WT KCNQ1+KCNE1
expressing cells, a small IKCNQ1-KCNE1was observed when the
atrial AP command was applied (Fig. 4Ai); current increased
relatively gradually during the applied AP command, attaining
its maximal amplitude relatively late during the AP. This is
consistent with a previous simulation of the profile of IKs
during atrial APs (28). For V307L KCNQ1+KCNE1, IKCNQ1-
KCNE1elicited by the atrial APwaveform was of a similar overall
profile to that of WT IKCNQ1-KCNE1 but exhibited a greater
magnitude (compare Fig. 4Ai with Fig. 4Aii). Fig. 4B shows
instantaneous I-V relations for WT and V307L during atrial AP
repolarisation (for the same cells as Fig. 4Ai and Aii): for 6
cells exhibiting WT IKCNQ1-KCNE1maximal repolarising current
occurred at -19.1±1.6 mV, whereas for 8 cells exhibiting
V307L IKCNQ1-KCNE1 this value was -20.9±1.0 mV (P>0.1 vs.
WT). As shown in Fig. 4C, the mean maximal amplitude of
V307L IKCNQ1-KCNE1during repolarisation was more than three-
fold that for WT IKCNQ1-KCNE1. However, for both WT and
mutant channels the current was much smaller than the
corresponding current seen during ventricular APs (compare
Fig. 3B,C and 4B,C). The atrial AP command differed from the
ventricular AP used (cf Fig. 4A with Fig. 3A) both in exhibiting
an initial rapid repolarisation phase and in possessing a lower
and briefer duration plateau phase. Consequently, both the
smaller current amplitude and the more negative value of peak
repolarising current can be attributed to the relatively lower
and shorter plateau phase of the atrial AP command, which
would result in less extensive current activation compared to
the ventricular AP waveform.
127
Fig. 4.WTand V307LIKCNQ1-KCNE1during atrial
AP clamp. (A) Profile of WT (Ai) and V307L
(Aii) IKCNQ1-KCNE1 (the thicker of the pair of
overlaid traces) during an applied atrial AP
command (the thinner of the pair of overlaid
traces; AP command frequency of 1 Hz). Each
of (Ai) and (Aii) has its own time axis, whilst
the current axis (shown to the left of (Ai)) and
voltage axis (shown to the right of (Aii)) apply
to both panels. (B) Representative
instantaneous current-voltage (I-V) relations
for WT (Bi) and V307L (Bii) IKCNQ1-KCNE1
during atrial AP repolarisation (direction of
repolarisation denoted by arrows).
Instantaneous I-V relations show continuous
plots of current recorded during AP
repolarisation and the direction of
repolarisation on each plot is indicated by the
filled 'arrow' symbols. (C) Comparison of mean
(±S.E.M) maximal repolarising current during
AP repolarisation between WT and V307L
IKCNQ1-KCNE1(n=6 and 8 respectively; P<0.05).