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The Journal of Neuroscience, August 1, 2001, 21(15):5535-5545
Activation of Expressed KCNQ Potassium Currents and Native
Neuronal M-Type Potassium Currents by the Anti-Convulsant Drug
Retigabine
L.
Tatulian1,
P.
Delmas2,
F. C.
Abogadie2, and
D. A.
Brown1
1 Department of Pharmacology and 2 Wellcome
Laboratory for Molecular Pharmacology, University College London,
London WC1E 6BT, United Kingdom
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ABSTRACT |
Retigabine [D-23129;
N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid
ethyl ester] is a novel anticonvulsant compound that is now in
clinical phase II development. It has previously been shown to enhance
currents generated by KCNQ2/3 K+ channels
when expressed in Chinese hamster ovary (CHO) cells (Main et al., 2000 ;
Wickenden et al., 2000). In the present study, we have compared the
actions of retigabine on KCNQ2/3 currents with those on currents
generated by other members of the KCNQ family (homomeric KCNQ1, KCNQ2,
KCNQ3, and KCNQ4 channels) expressed in CHO cells and on the native M
current in rat sympathetic neurons [thought to be generated by KCNQ2/3
channels (Wang et al., 1998)]. Retigabine produced a hyperpolarizing
shift of the activation curves for KCNQ2/3, KCNQ2, KCNQ3, and KCNQ4
currents with differential potencies in the following order: KCNQ3 > KCNQ2/3 > KCNQ2 > KCNQ4, as measured either by the
maximum hyperpolarizing shift in the activation curves or by the
EC50 values. In contrast, retigabine did not enhance
cardiac KCNQ1 currents. Retigabine also produced a hyperpolarizing
shift in the activation curve for native M channels in rat sympathetic
neurons. The retigabine-induced current was inhibited by muscarinic
receptor stimulation, with similar agonist potency but 25% reduced
maximum effect. In unclamped neurons, retigabine produced a
hyperpolarization and reduced the number of action potentials produced
by depolarizing current injections, without change in action potential configuration.
Key words:
potassium channels; KCNQ channels; M channels; sympathetic neurons; retigabine; anti-convulsant
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INTRODUCTION |
M-type potassium
(K+) currents
(IK(M)) are a species of subthreshold
voltage-gated K+ current that serve to
stabilize the membrane potential and control neuronal excitability.
They were first described in sympathetic neurons but have subsequently
been reported in various other neurons, including hippocampal and
cortical pyramidal cells (for review, see Brown, 1988; Marrion,
1997).
Recent evidence suggests that the native M channels in rat sympathetic
neurons are composed of a heteromeric assembly of KCNQ2 and KCNQ3
K+ channel subunits (Wang et al., 1998;
Hadley et al., 2000; Selyanko et al., 2000b; Shapiro et al., 2000).
These are K+ channel gene products that
are widely distributed in the nervous system, mutations of which give
rise to a form of congenital epilepsy termed benign
familial neonatal convulsions (Biervert et al., 1998; Charlier et al.,
1998; Singh et al., 1998; for review, see Rogawski, 2000; Jentsch,
2000). This implies that M channels may assist in controlling seizure
discharges and that drugs that enhance M channel activity might be
effective anti-epileptic agents. In accordance with this, the
anti-convulsant drug retigabine [D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid
ethyl ester] (Rostock et al., 1996; Tober et al., 1996) has been
reported to open K+ channels in NG108-15
neuroblastoma-glioma hybrid cells (Rundfeldt, 1997) and to enhance
currents through expressed heteromeric KCNQ2/3 channels, in part by
shifting their voltage sensitivity to more hyperpolarized membrane
potentials (Main et al., 2000; Rundfeldt and Netzer, 2000;
Wickenden et al., 2000).
Two questions arise, however, concerning the effect of
retigabine. First, KCNQ2 and KCNQ3 are members of a larger family of homologous K+ channels comprising, in
addition, a subunit (KCNQ1) of the cardiac delayed rectifier current
(Barhanin et al., 1996; Sanguinetti et al., 1996), a subunit (KCNQ4)
present in the auditory system (Kubisch et al., 1999; Kharkovets et
al., 2000), and another recently identified subunit (KCNQ5) widely
distributed in the CNS and PNS (Lerche et al., 2000; Schroeder et al.,
2000). All of these subunits, when expressed as homomultimers, generate
"M-like" currents as defined kinetically and pharmacologically
(Hadley et al., 2000; Selyanko et al., 2000b; Schroeder et al.,
2000). It is not yet known whether they are all equally sensitive to
retigabine. Second, it has yet to be established whether retigabine
affects native neuronal M currents in the same way that it affects
expressed KCNQ2/3 channels. Accordingly, in the present experiments, we have compared the action of retigabine on heteromeric KCNQ2/3 currents
expressed in mammalian CHO cells with its action on expressed homomeric
KCNQ1, KCNQ2, KCNQ3, and KCNQ4 currents, on the one hand, and on native
M currents in rat sympathetic neurons on the other.
While this work was in progress, Wickenden et al. (2001) reported that
retigabine also enhances currents through concatameric KCNQ3/5 channels
in CHO cells.
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MATERIALS AND METHODS |
Chinese hamster ovary cell culture and transfection.
Chinese Hamster Ovary (CHO) cells stably transfected with cDNA encoding human M1 muscarinic receptors (Mullaney et al.,
1993) were cultured in  -MEM supplemented with 10% fetal calf serum,
1% L-glutamine, and 1% penicillin/streptomycin. Cells
were split when confluent and plated onto 35 mm dishes. Cells were left
at 37°C and 5% CO2 for 1 d before
transfection using LipofectAmine Plus (Life Technologies, Gaithersburg,
MD) according to the manufacturer's recommendations. Plasmids
containing KCNQ and CD8 cDNAs, and driven by the cytomegalovirus promoter, were cotransfected in a ratio of 10:1. For expression of
heteromultimers, equal amounts of KCNQ2 and KCNQ3 cDNAs were used.
Successfully transfected cells were identified by adding CD8-binding
Dynabeads (Dynal, Great Neck, NY) before electrophysiological recording.
Ganglion cell culture. Primary cultures of neurons were
prepared from superior cervical ganglia (SCG) from 17-d-old Sprague Dawley rats, using a standard enzymatic dissociation procedure, as
fully described elsewhere (Delmas et al., 1998b). Rats were killed by
CO2 inhalation and decapitated, according to the
U.K. Animals (Scientific Procedures) Act 1986. Ganglia were dissected from the carotid bifurcation, and the surrounding sheath was removed. Each ganglion was cut into several pieces with iridectomy scissors and
incubated at 37°C in collagenase (500 U/ml and bovine serum albumin 6 mg/ml) and then in trypsin (1 mg/ml with 6 mg/ml BSA). After
trituration the cells were centrifuged, and the pellet was resuspended
in culture medium (L-15 medium supplemented with 10% fetal bovine
serum, 24 mM NaHCO3, 38 mM glucose, 50 U/ml penicillin and streptomycin,
25 ng/ml nerve growth factor). After the dissociated cells were plated
onto laminin-coated 35 mm plastic Petri dishes, the cells were kept in
a 37°C incubator with 5% CO2.
Electrophysiological recording. Currents in CHO cells and
SCG neurons were recorded using the amphotericin-B perforated-patch technique (Rae et al., 1991). Patch electrodes (2-3 M ) were filled by dipping the tip for 40 sec into the appropriate filtered internal solution, and the pipette was then backfilled with the internal solution containing 0.1-0.2 mg/ml amphotericin-B. After
permeabilization, access resistances were generally <15 M. The
recording electrodes had resistance of 2-3 M when filled with
internal solution.
Recordings from CHO cells were made at room temperature 1-2 d after
transfection. No appreciable current was noticed in untransfected CHO
cells. The extracellular solution consisted of (in mM):
NaCl 144, KCl 2.5, CaCl2 2, MgCl2 0.5, HEPES 5, and glucose 10; pH 7.4 with
Tris base. To record tail currents at  120 mV (for the purpose of
construction of activation curves), external KCl was raised to 25 mM; the increase in osmolarity was compensated by reducing
the concentration of NaCl to 121.5 mM. The internal
(pipette) solution contained (in mM):
K+ acetate 80, KCl 30, HEPES 40, MgCl2 3, EGTA 3, CaCl2 1;
pH 7.4 with KOH.
K+ and Ca2+
current recordings in SCG neurons were as described (Delmas et al.,
2000). Briefly, SCG neurons were perfused with an external solution
consisting of (in mM): NaCl 120, KCl 3, HEPES 5, NaHCO3 23, glucose 11, MgCl2 1.2, CaCl2 2.5, tetrodotoxin 0.0005, pH 7.4. Internal solution for M-current
recording consisted of (in mM):
K+ acetate 90, KCl 40, HEPES 20, MgCl2 3 (adjusted to pH 7.3-7.4 with KOH, and
300 mOsm/l with K+ acetate); for
Ca2+ current recording the solution
consisted of (in mM): CsCl 30, cesium acetate 110, HEPES
10, MgCl2 1 (pH 7.2-7.3 with CsOH, 300 mOsm/l).
Experiments were performed at 30-32°C.
Voltage-clamp recordings. Data were acquired and analyzed
using pClamp software (version 6.0.3). Currents in transfected CHO cell
line were recorded using Axopatch 1D patch-clamp amplifier (Axon
Instruments) and filtered at 1 kHz. The capacity transients were
cancelled using the resistance capacitance circuit within the
amplifier. Series resistance compensation was set to 60-80%.
SCG neurons were voltage clamped using an Axopatch 200A amplifier (Axon
Instruments). Current traces were low-pass filtered at 2-5 kHz using a
four-pole Bessel filter, and series resistance and membrane capacitance
were partially compensated (>70%). Leak and capacitance currents were
subtracted digitally using the P/6 subtraction procedure of Pclamp6.
The capacity transients were cancelled using the resistance capacitance
circuit within the amplifier. Series resistance compensation was also
used and was set to 60-80%.
The data were analyzed using the Clampfit (pClamp6 and 7), ORIGIN 5, and Excel data handling and graphical presentation software packages.
The methods of recording and analysis were similar to those used
previously for studying KCNQ currents (Selyanko et al., 2000b). Results
are presented as the mean ± SEM.
Activation curves were fitted by the Boltzmann equation:
![<FR><NU>I</NU><DE>I(50)</DE></FR>=<FR><NU>1</NU><DE>[1+<UP>exp</UP>((V<SUB>1/2</SUB>−V)/k)]</DE></FR>,](/content/vol21/issue15/fulltext/5535/img001.gif)
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(1)
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where I is the tail current recorded at 120 mV
after a pre-step to membrane potential V (estimated from the amplitudes
of exponentials backfitted to the beginning of the test step),
I(50) is the current after a step to +50 mV,
V1/2 is the membrane potential at
which I is equal to one-half I(50), and
k is the slope of the curve. The concentration-response of
different KCNQ channels to retigabine was estimated by the shift in the
V1/2 values caused by 0.1, 0.3, 1, 3, 10, 30, 100, and 300 µM. Dose-response curves were fitted to the following equation:
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(2)
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where EC50 is the concentration
corresponding to half-maximal activity, x is the agonist
concentration, and p is the slope of the curve.
Inhibition of the KCNQ1 current was evaluated by the decrease in
current amplitude after step depolarizations to +50 mV from 70 mV
after addition of 3, 10, 30, 100, 300, and 1000 µM of
retigabine. The inhibition curve was fitted with Equation 2, with
IC50 concentration corresponding to half-maximal block.
Chemicals and drugs. cDNAs for human KCNQ2 and rat KCNQ3
were obtained from Dr. D. McKinnon (Department of Neurobiology and Behavior, State University of New York, Stony Brook, NY), for human
KCNQ1 from Dr. M. T. Keating (Howard Hughes Medical
Institute, University of Utah, Ogden, UT), and for KCNQ4
from Dr. T. J. Jentsch (Zentrum für Molekulare
Neurobiologie, Hamburg, University of Hamburg, Hamburg,
Germany). Retigabine was obtained from Glaxo SmithKline (Stevenage,
UK). Oxotremorine methiodide (Oxo-M) was obtained from RBI (Natick,
MA). All other drugs and chemicals were obtained from Sigma or BDH
(Poole, UK).
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RESULTS |
Retigabine enhances KCNQ2-4 currents in CHO cells
Figure 1 illustrates the effect of
10 µM retigabine on currents carried by heteromeric
KCNQ2/3 channels and homomeric KCNQ2, KCNQ3, and KCNQ4 channels
expressed in CHO cells. In these experiments, an "M-current
protocol" (Adams and Brown, 1982) was used to record the expressed
currents; that is, the cell was predepolarized to 20 mV to activate
the current, then hyperpolarized to 90 mV in steps of 10 mV to
deactivate the current. Deactivation is registered by the slow tail
currents, and reactivation is registered on stepping back to 20 mV by
the slow outward current. The following points emerge from this
comparison. (1) No appreciable current could be recorded from control
(untransfected) cells, and retigabine had no significant effect on
these cells (Fig. 1B). (2) As predicted from the
previous experiments of Main et al. (2000), Rundfeldt and Netzer
(2000), and Wickenden et al. (2000), retigabine increased the outward
current recorded at 20 mV in cells transfected with KCNQ2 + KCNQ3
cDNAs. However, it also increased the 20 mV current in cells
transfected solely with KCNQ2, KCNQ3, or KCNQ4 cDNAs to comparable (or
greater) extents. (3) The additional current at 20 mV induced by
retigabine in KCNQ2- and KCNQ2/3-transfected cells was removed by
hyperpolarizing to 90 mV. However, an additional component of inward
current was recorded at 90 mV in KCNQ3- and KCNQ4-transfected cells.
This implies that a component of KCNQ3 or KCNQ4 current persisted in
the presence of retigabine negative to resting potentials (60 mV)
(Selyanko et al., 2000b), where these channels would normally be fully
deactivated. (4) As also reported previously for KCNQ2/3 currents (Main
et al., 2000; Rundfeldt and Netzer, 2000; Wickenden et al., 2000) and
KCNQ3/5 currents (Wickenden et al., 2001), retigabine slowed the
deactivation of the currents during step-hyperpolarizations and
accelerated the reactivation on repolarization. This effect was most
pronounced with KCNQ3, for which the time dependence of current
deactivation was lost except at very negative potentials, and the
effect was least pronounced for KCNQ4. Thus, these tests indicate that
the previously reported enhancing effect of retigabine on KCNQ2/3 and
KCNQ3/5 channels also extends to homomeric KCNQ2, KCNQ3, and KCNQ4
channels, albeit with quantitative differences (see further below).
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Figure 1.
Enhancement of heteromeric
(KCNQ2/3) and homomeric (KCNQ2-4)
currents in CHO cells by retigabine. A, Voltage
protocol: currents were activated by clamping the membrane at 20 mV
and then deactivated by 1 sec hyperpolarizations to 90 mV in 10 mV
increments. B, Retigabine (10 µM) did not
activate any endogenous currents in untransfected CHO cells.
C, KCNQ currents generated by the voltage protocol in
A recorded in the absence (left),
presence (center), and 10 min after washout
(right) of retigabine. Dotted lines
denote zero current level.
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Augmentation of KCNQ2-4 currents by retigabine was confirmed after
current activation from 70 mV to +50 mV in 10 mV steps (Figs.
2, 3).
Several points are worth noting. First, retigabine clearly accelerated
the onset of all currents, but most noticeably with KCNQ3. Second,
current-voltage curves were shifted to the left (Fig. 3). For KCNQ2,
KCNQ4, and KCNQ2/3, this corresponded to a ~20-30 mV negative shift
in threshold for current activation. For KCNQ3 the shift was much
greater because the current was still active at the holding potential
(70 mV): in effect, retigabine converted the KCNQ3 current from a
time- and voltage-dependent current with a threshold of 50 mV to a
substantially time- and voltage-independent "leak" current from
90 mV upward. Third, retigabine clearly increased the maximal current
generated by KCNQ3 and KCNQ4 channels (and to varying extents, by
KCNQ2/3 channels) but not by KCNQ2 channels. Variation in the amount by
which retigabine increased the maximum current amplitude through
different KCNQ channels may reflect a variable component of secondary
blocking action at positive potentials (see KCNQ1 below).
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Figure 2.
Activation of heteromeric KCNQ2/3 and homomeric
KCNQ2-4 channels expressed in CHO cells in the absence and presence of
retigabine. Families of KCNQ currents were recorded in the absence
(left) or presence (right) of 10 µM retigabine. Currents were activated by 1 sec
depolarizing pulses in 10 mV increments to +50 mV from a holding
potential of 70 mV. Records show currents recorded at 40, 20, 0, and +20 mV. Note that KCNQ3 and KCNQ4,
but not KCNQ2/3 and KCNQ2 currents, are
increased at all voltages by retigabine.
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Figure 3.
Effects of retigabine on the KCNQ2-4
current-voltage relationship. Normalized current values were plotted
against command potential before ( ) and after ( ) addition of 10 µM retigabine. To obtain normalized values, peak current
amplitudes in response to depolarizing pulses from a holding voltage of
70 to +50 mV in 10 mV increments were normalized against the maximum
current amplitude at +50 mV in control recordings. Retigabine shifts
the threshold for channel activation and augments KCNQ current
amplitude across a range of membrane potentials.
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Retigabine shifts KCNQ activation curves
The current-voltage curves in Figure 3 suggest that one effect of
retigabine is to produce a negative shift in the KCNQ current activation curve. This was further tested using the protocol shown in
Figure 4, in which the current was first
fully activated by stepping to +50 mV, then stepped for 1 sec to
various potentials between +50 and 110 mV, followed by a final step
to 120 mV (Fig. 4A). The relative amount of
conductance activated at each potential at the end of the 1 sec step
could then be determined directly from the residual tail-current at
120 mV. Boltzmann plots (Fig. 4B) (see Materials
and Methods) confirmed that 10 µM retigabine produced a significant left-shift of the activation curves in the order
KCNQ3 (43 mV) > KCNQ2/3 (30 mV) > KCNQ2 (24 mV)
>KCNQ4 (14 mV). (Values in brackets give mean shift in
half-activation potential V1/2.) In
contrast, the slopes of the activation curves were not significantly
changed by retigabine (Fig. 4, legend).
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Figure 4.
Effects of retigabine on the KCNQ2-4 activation
curves. A, Currents were activated by depolarizing
pulses to +50 mV from a holding potential of 70 mV and deactivated by
1 sec hyperpolarizing pulses to various potentials between +50 and
120 mV, followed by a step to 120 mV. The
inset shows the tail currents recorded at 120 mV,
which were used to construct the activation curves. B,
Activation curves in the absence () and presence () of retigabine
were obtained from tail currents as described in Materials and Methods
and fitted with the Boltzmann Equation 1. The
V1/2 values were as follows: KCNQ2/3,
17.3 ± 2.2 mV for control (n = 9) and
47.7 ± 2.7 mV for retigabine-treated cells
(n = 9); KCNQ2, 11.5 ± 1.5 mV for
control (n = 16) and 35.7 ± 2.1 mV for
retigabine-treated cells (n = 6); KCNQ3,
28.7 ± 2.5 mV for control (n = 7) and
71.5 ± 3.1 mV for retigabine-treated cells
(n = 7); and KCNQ4, 12.6 ± 0.7 mV for
control (n = 4) and 26.2 ± 0.6 mV for
retigabine-treated cells (n = 4). Slopes were as
follows: KCNQ2/3, 12.9 ± 1.2 mV (control, n = 9) and 11.4 ± 1.3 mV (retigabine, n = 5);
KCNQ2, 10.9 ± 0.6 mV (control, n = 16) and
11.2 ± 0.9 mV (retigabine, n = 6); KCNQ3,
11.4 ± 0.9 mV (control, n = 7) and 10.9 ± 0.9 mV (retigabine, n = 7); and KCNQ4, 10.1 ± 0.3 mV (control, n = 4) and 12.1 ± 1.6 mV
(retigabine, n = 4). The data shown are mean ± SEM.
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The differential effects shown in Figure 4 were obtained using a fixed
(10 µM) concentration of retigabine. To measure the potency of retigabine, the shift in the activation curves produced by
incremental concentrations of retigabine were determined, as illustrated in Figure 5. To amplify the
inward tail currents at 120 mV (and hence improve the accuracy of the
deduced activation curves), the external
K+ concentration was raised to 25 mM for these experiments. This did not itself affect the
action of retigabine because the shift of the KCNQ2/3 activation curve
produced by 10 µM retigabine in 25 mM
[K+] was indistinguishable from that
observed in 2.5 mM [K+],
which is illustrated in Figure 4. The magnitude of the shift in the
activation curves was clearly dependent on the concentration of
retigabine (Fig. 5B). The relation between the concentration of retigabine and the shift in the half-activation potential
V1/2 for the different KCNQ channels
tested deduced from these experiments, and normalized to the maximum
shift produced by 10-300 µM retigabine, is
shown in Figure 6. Data points could be
fitted with a Hill equation (see Materials and Methods), with slope
~1 but EC50 values varying from 0.6 µM for KCNQ3 to 5.2 µM
for KCNQ4. The maximum shift of V1/2
also varied with different KCNQ channels. The order of potency as
determined from EC50 values (KCNQ3 > KCNQ2/3 > KCNQ2 > KCNQ4) accords with the apparent
"efficacy" as measured by the maximum shift (Table
1).
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Figure 5.
Effects of retigabine on KCNQ currents are
concentration dependent. A, Experiments were performed
in high extracellular K+ (25 mM)
solution to enhance the tail currents. A family of currents for KCNQ2/3
and the voltage protocol under these conditions are shown. Application
of retigabine led to a slowing in the rate of decline of the KCNQ tail
current (inset). B, Representative
activation curves for KCNQ2/3 and KCNQ4 generated in the absence ( )
and presence of 1 µM ( ), 3 µM ( ), 10 µM (), 30 µM ( ), 100 µM
( ), and 300 µM ( ) retigabine. For KCNQ4, 1 mM retigabine ( ) was also applied.
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Figure 6.
Concentration-response curves for KCNQ2/3, KCNQ2,
KCNQ3, and KCNQ4. The magnitude of the leftward shift in the
half-activation potential (V1/2) was
calculated with each concentration of retigabine and normalized against
the maximum shift. The maximum shift in the activation curve
was obtained with 10 µM retigabine for
KCNQ2/3, KCNQ2, and KCNQ3 and with 300 µM for KCNQ4.
Normalized values were plotted against retigabine concentration, and
the data were fitted with Equation 2 with the following parameters:
EC50 = 1.9 ± 0.2 µM and slope = 1.3 ± 0.2 for KCNQ2/3 (n = 5);
EC50 = 2.5 ± 0.6 µM
and slope = 1.1 ± 0.5 for KCNQ2
(n = 5); EC50 = 0.6 ± 0.3 µM and slope = 1.2 ± 0.4 for KCNQ3
(n = 3); and EC50 = 5.2 ± 0.9 µM and slope = 0.9 ± 0.2 for KCNQ4
(n = 3). Each point represents the mean ± SEM.
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Interaction of retigabine and linopirdine on KCNQ2/3 currents
The blocking action of the M-channel antagonist linopirdine (Lamas
et al., 1997) on KCNQ2/3 currents was unaffected by the presence of
retigabine. IC50 values were 7.3 ± 1.7 µM (n = 3) in the absence of retigabine
and 11.1 ± 2.3 µM (n = 4)
in the presence of 10 µM retigabine. These
values were not significantly different (p = 0.27; t test). The IC50 value in the
absence of retigabine is not dissimilar from that (4.0 ± 0.5 µM) reported previously by Wang et al. (1998)
against KCNQ2/3 currents expressed in frog oocytes.
KCNQ1 currents are resistant to enhancement by retigabine
In contrast to its effects on KCNQ2-4 currents, retigabine (10 µM) did not enhance currents generated by homomeric KCNQ1
channels in CHO cells (Fig.
7A) nor did it shift the KCNQ1
activation curve (Fig. 7C). Instead, at positive potentials,
higher concentrations of retigabine (100 µM)
reduced the KCNQ1 current amplitude in an apparently voltage-dependent
manner, the fractional reduction increasing with increasing positivity
(Fig. 7B,D). This effect was even
more pronounced at 1 mM. The blocking action of
retigabine on KCNQ1 current was quantitated by applying 3, 10, 30, 100, 300, and 1000 µM retigabine and measuring the
reduction of current at +50 mV. Fractional inhibition of the current
was plotted against retigabine concentration and points fitted with a
Hill equation (see Materials and Methods), with a slope of 1.26 ± 0.16 and IC50 of 100.1 ± 6.5 µM (n = 4) (Fig. 7E)
(note that only ~65% of the KCNQ1 current was inhibited with 1 mM retigabine). Retigabine had similar effects
(no activation shift and partial current block at positive potentials)
on currents generated by coexpressed KCNQ1 + KCNE1 channels
(n = 4). A variable expression of the voltage-dependent inhibitory action of retigabine revealed in KCNQ1 channels may account
for the variable degree of current enhancement at positive potentials
through different KCNQ channels illustrated in Figure 2.
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Figure 7.
Retigabine does not enhance KCNQ1 current in CHO
cells. A, KCNQ1 currents were recorded using the
protocol shown in inset in the absence
(left) and presence (right) of 10 µM retigabine. B, Normalized
current-voltage curve for KCNQ1 in the absence () and presence
() of 100 µM retigabine. Voltage protocol as in Figure
2. Note the reduction of peak current in the presence of retigabine.
C, Activation curves were constructed for KCNQ1 in the
absence (, n = 11) and presence (,
n = 5) of 100 µM retigabine as
described in Figure 4. Lines are Boltzmann fits to the data, giving
V1/2 = 12.5 ± 1.1 mV and slope
of 12.4 ± 0.9 mV for control and
V1/2 = 13.2 ± 1.2 mV and slope
of 12.4 ± 0.9 mV in the presence of retigabine.
V1/2 values are not significantly different
(unpaired t test; p = 0.7).
D, Voltage dependence of inhibition: fraction of current
inhibited by 100 µM retigabine plotted against voltage
(replotted from B). E, Concentration
dependence of inhibition. Fractional inhibition at +50 mV
(ordinate) was plotted against log M retigabine
concentration (abscissa). Data were fitted with Equation 2 (see Materials and Methods). Points show means ± SEM, with
n = 4. Mean IC50 was 100.1 ± 6.5 µM, and the slope was 1.26 ± 0.16.
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Retigabine enhances native M currents in sympathetic neurons and
shifts the voltage dependence for their activation
The native M current in rat SCG neurons has been attributed to
currents carried through heteromeric KCNQ2/3 channels (Wang et al.,
1998). Hence, retigabine might be expected to affect these native M
currents in a manner resembling its effect on KCNQ2/3 channels. In the
present experiments we therefore recorded native M currents in
dissociated rat SCG neurons using the perforated-patch configuration of
the patch pipette (see Materials and Methods).
Figure 8 illustrates the effect of 10 µM retigabine on the outward currents evoked in a
dissociated rat SCG neuron by 1 sec depolarizing steps from 80 mV. In
the absence of retigabine, activation of M current is manifest by the
appearance of a slow time-dependent component of outward current at
command potentials of 50 mV and upward, subsequent to the transient
(~50-100 msec duration) "A-current" (Fig. 8A).
In the presence of retigabine, two differences are seen: the
time-dependent component appears at a more negative command potential
(70 mV), and the total outward current is increased at all command
potentials. The extra current induced by retigabine is indicated by the
subtracted currents in Figure 8B: this current is
clearly "M-like" in appearance, with slow activation and
deactivation, and saturates at 30 mV. Note that unlike the raw
currents, the subtracted current is devoid of an initial transient
A-current component, implying that the A-current was not enhanced by
retigabine.
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Figure 8.
Retigabine enhances outward currents in SCG
neurons. A, Families of currents recorded from a rat SCG
neuron elicited by 1 sec voltage steps from a holding potential of 80
mV to test potentials from 70 to 10 mV (as indicated). Retigabine
(RTG) was applied via bath perfusion at 10 µM. b, Retigabine-induced outward currents
obtained by subtracting control currents from currents in the presence
of retigabine. Note the saturation of the current at potentials
positive to 30 mV.
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The effect of retigabine on the current-voltage curves for the native
M current was further examined by applying slow voltage ramps in the
presence and absence of the M current blocker linopirdine (10 µM) (Fig.
9A). In this way, the
component of outward rectification in the current-voltage curves
attributable to M current could be identified as that component of
current blocked by linopirdine (Fig. 9B). In the presence of
retigabine, this component of rectification was shifted ~20 mV in the
negative direction (mean, 21 ± 2 mV; n = 5). As
a result, the threshold for the outwardly rectifying M current was
shifted from between 50 and 60 mV to approximately 80 mV.
However, a small component of linopirdine-sensitive outward current,
which appeared insensitive to voltage, could also be detected at
potentials negative to 80 mV in the presence of retigabine. This was
confirmed in the form of a persistent inward current on raising
external [K+] to 20 mM (Fig. 9C). As with expressed
KCNQ2/3 and KCNQ2 channels (Fig. 3), retigabine did not increase the
maximum amplitude of the linopirdine-sensitive component of current.
Thus, the effects of retigabine on the linopirdine-sensitive (presumed
M) component of current, a negative shift in the current-voltage curve
without increased maximum, were in qualitative agreement with its
effects on expressed KCNQ2/3 channels. (Although the concentration of linopirdine used in these experiments was unlikely to have completely blocked the M current, the fraction blocked was presumably the same in
the absence and presence of retigabine, because retigabine did not
affect the action of linopirdine on expressed KNCQ2/3 currents; see
above.)
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Figure 9.
Retigabine shifts the voltage dependence of
activation of the M current. A, Superimposed
steady-state I-V relationships
(determined using a voltage ramp from 100 to 0 mV at 5 mV/sec) from a
single cell in control conditions and after addition of linopirdine (10 µM), retigabine (10 µM, after washout of
linopirdine), and retigabine + linopirdine. B,
Difference currents obtained by digitally subtracting the
I-V relationships shown in
A. Note the leftward shift in the activation of the M
current (linopirdine-sensitive current) induced by retigabine and the
bell shape of the retigabine induced current. C,
Retigabine-induced current in the presence of 3 and 20 mM
external K+. Calculated
EK values were 102 and 56 mV,
respectively.
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Enhancement of M current in SCG neurons was confirmed using the
standard M-current protocol, in which M current is recorded in
isolation from inactivating currents by predepolarizing to 20 mV and
then deactivating the M current by step hyperpolarization to 50 mV
(Fig. 10; compare Fig. 1). Incremental
addition of increasing concentrations of retigabine produced a
progressive increase in the steady outward current at 20 mV, with an
EC50 of ~0.76 µM (Fig.
10B) (mean 0.74 ± 0.02 µM; n = 5). There was also an
increase in the residual SCG M current at 50 mV, implying that the
hyperpolarizing step no longer fully deactivated the M current (as
expected from the negative shift of the activation curve). These
experiments also revealed that retigabine slowed the deactivation of
the M current at 50 mV, the time constant for the faster component of
the relaxation being slowed from ~30 to ~80 msec (Fig.
11).
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Figure 10.
Modulation of the M current by retigabine is
concentration dependent. A, Superimposed M-current
traces before and after cumulative addition of retigabine (0.1-30
µM). The M current was recorded in response to 1 sec
hyperpolarizing steps from a holding potential of 20 to 50 mV
followed by a ramp to 90 mV. Note that the increase in holding
current is associated with alteration of M-current deactivation
relaxation. B, Holding current at 50 and 20 mV
plotted against retigabine concentrations for the cell illustrated in
A. Solid lines represent best fits to the
Hill equation with holding currents in the absence of retigabine of 610 pA at 20 mV and 42 pA at 50 mV (as indicated by
arrows). EC50 values and Hill coefficient
(slope) are indicated. Mean values are given in Results.
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Figure 11.
Muscarinic inhibition of retigabine-enhanced M
current. A, Currents were produced by holding at 20 mV
and giving repeated steps to 80 mV in 10 mV increments. Steady-state
currents were enhanced by retigabine (RTG) and inhibited
by oxotremorine-M (Oxo-M). The
inset shows currents induced by the simultaneous
application of retigabine and Oxo-M. B, Families of
M-current deactivations obtained before (Control)
and after addition of retigabine (10 µM) and retigabine
(10 µM) + Oxo-M (10 µM).
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The retigabine-enhanced M current is sensitive to inhibition by
muscarinic receptor agonists
The native M current in rat SCG neurons is inhibited by
stimulating endogenous M1 muscarinic receptors
(Marrion et al., 1989; Bernheim et al., 1992). We therefore tested
whether the retigabine-modified M current was also sensitive to
muscarinic receptor stimulation. As shown in Figure 11, this appeared
to be the case: retigabine (10 µM) augmented the outward
current recorded at 20 mV, and subsequent addition of the muscarinic
receptor agonist Oxo-M (10 µM) eliminated this
extra current. The inhibitory effect of Oxo-M was quantitated by
measuring the initial amplitude of the deactivation tail currents at
50 mV recorded in the presence of incrementing concentrations of
Oxo-M in the absence and presence of retigabine. As indicated in Figure
12A, the
IC50 values for Oxo-M were similar in the
presence and absence of retigabine. However, the maximum inhibition
produced by Oxo-M (30 µM) was reduced by
~25%. This was unlikely to have been caused by an effect
(noncompetitive) of retigabine on the muscarinic receptors because
retigabine did not reduce the effect of Oxo-M on the
Ca2+ current recorded in pertussis
toxin-treated cells (Fig. 12B), which is mediated by
M1 receptor activation (Bernheim et al., 1992;
Delmas et al., 1998b). Hence, although equally sensitive to Oxo-M in
terms of IC50 values, retigabine-modified M
currents appear somewhat less susceptible to muscarinic receptor
modulation in terms of the amount of inhibition. One additional point
emerging from these experiments was that the current-enhancing effect
of retigabine was more rapid in onset than the current-inhibiting effect of Oxo-M when the two were applied simultaneously (Fig. 11A, inset). This implies that retigabine
has a more direct effect on the M channels than Oxo-M, the action of
which involves a diffusible second messenger (Selyanko et al.,
1992).
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Figure 12.
Retigabine-enhanced M currents are more resistant
to modulation. A, Concentration dependence of M-current
inhibition by Oxo-M in the presence () or absence () of
retigabine (10 µM). B, Concentration
dependence of Ca2+ current inhibition by Oxo-M in
the presence () or absence () of retigabine (10 µM). Ca2+ currents were recorded using
the perforated-patch method in cells pretreated with pertussis toxin
(0.5 µg/µl) for 24 hr (Delmas et al., 1998a). Points were fitted
with Equation 2 (see Materials and Methods).
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Selectivity of retigabine action on rat SCG neurons
As pointed out in connection with Figure 8 above, retigabine (10 µM) had no significant effect on the transient
"A-type" current recorded in rat SCG neurons (n = 12), which is probably mediated by Kv4.2
K+ channels (Malin and Nerbonne, 2000).
Likewise, 10 µM retigabine did not affect the
hyperpolarization-activated cation current Ih (n = 4) (Lamas,
1998) or the N-type Ca2+ current
(n = 6). However, it did exert a small (20 ± 2%;
n = 4) inhibitory effect on the
Ca2+-activated
K+ current driving the post-spike
after-hyperpolarization.
Physiological consequences of retigabine action
Because the M current acts as a braking current on action
potential discharges (Brown, 1988), retigabine might be expected to
reduce spike activity. Figure 13
illustrates a test for this on an SCG neuron. In Figure 13A,
the neuron was challenged with 0.5 sec depolarizing or hyperpolarizing
current injections from a resting membrane potential of approximately
60 mV. The depolarizing pulses typically evoked a phasic discharge of
one or two spikes. The application of retigabine (3 µM) hyperpolarized the cells by 9 ± 4 mV (n = 7). When the membrane potential was brought back to 60 mV, the original depolarizing current injections no longer
induced spikes (Fig. 13B). This occurs because the increase of M current conductance exerts a more effective brake on firing, as
evidenced by the appearance of hyperpolarizing sags during depolarization (Fig. 13B). Retigabine also decreased the
voltage response to hyperpolarizing current pulses, as expected from an increase in cell conductance, but had no effect on action potential size or shape (Fig. 13D).
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Figure 13.
Retigabine abolishes firing in SCG neurons.
Voltage responses to depolarizing and hyperpolarizing current pulses
recorded from an SCG neuron before (A) and during
the application of retigabine (RTG; 3 µM)
(B, C). Note in C that
larger depolarizing current pulses are required to evoke an action
potential in the presence of retigabine. D, Action
potential evoked by a 1 msec/700 pA depolarizing pulse in the presence
(thick line) and absence (thin line) of
retigabine (3 µM).
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DISCUSSION |
The present experiments show that the previously described
property of retigabine to enhance K+
currents through heteromeric KCNQ2/3 channels (Main et al., 2000; Rundfeldt and Netzer, 2000; Wickenden et al., 2000) and KCNQ3/5 channels (Wickenden et al., 2001) also extends to currents through homomeric KCNQ2 and KCNQ3 channels, and through the homologous KCNQ4
channels. Available information suggests that the homomeric KCNQ5
channels (Lerche et al., 2000; Schroeder et al., 2000) are also
sensitive to retigabine (B. Jensen, personal communication). In
contrast, the cardiac KCNQ1 channels, expressed either homomerically or
as heteromeric assemblies with their natural cardiac subunit KCNE1,
appear to be insensitive to the current-enhancing action of retigabine.
Hence, current enhancement by retigabine is confined to neurally
expressed KCNQ channels.
In agreement with Wickenden et al. (2000, 2001), we find that the
principal action of retigabine is to produce a shift in the KCNQ
activation curve to more hyperpolarized potentials. The maximum shift
varied with different KCNQ channels in the order KCNQ3 > KCNQ2/3 > KCNQ2 > KCNQ4; the potency of retigabine, as measured by the concentration required to produce a half-maximal shift,
also followed the same order. As noted previously for KCNQ2/3 and
KCNQ3/5 channels (Main et al., 2000; Wickenden et al., 2000, 2001),
this shift is accompanied by a slowing of current deactivation and
acceleration of current activation. To an extent, this would be
anticipated from the initial descriptions of M-current kinetics (Adams
and Brown, 1982) if the channels "sensed" a progressively more
hyperpolarized potential. However, this interpretation may be too
simple, because the change in kinetics does not quantitatively match
that predicted from the original kinetic scheme for a given shift in
steady-state activation. M-channel kinetics are clearly more complex
than originally envisaged (Marrion, 1997; Selyanko and Brown, 1999),
and the kinetics of KCNQ channels per se have not yet been analyzed in
sufficient detail to suggest a more precise interpretation of the
action of retigabine.
At strongly positive potentials, the action of retigabine on KCNQ
channels appears to be complicated by a secondary inhibitory action.
This is most clearly seen with KCNQ1 channels, in which there is no
voltage shift and hence no current enhancement (Fig. 7), but it
probably extends to KCNQ2 and KCNQ2/3 channels, thereby accounting for
the limited increase in maximal current through these channels and
hence providing some degree of "self-limitation" to the enhancement
of currents through these channels. [The maximum Popen through KCNQ2 and KCNQ2/3
channels expressed in CHO cells is well below unity when recorded in
the absence of retigabine (Selyanko et al., 2000a; our
unpublished observations) and hence does not preclude an increase in
maximum current through these channels in the presence of retigabine.]
Because the IC50 for inhibition of KCNQ1 channels
(~100 µM) is between 20 and 100 times greater
than the EC50 for enhancement of KCNQ2-4
currents, this seems unlikely to provide a major constraint against the
potential therapeutic applications of retigabine.
A second important point established in the present experiments is that
retigabine also enhances currents through the native M channels in rat
sympathetic neurons. As with KCNQ channels, this is caused primarily by
a hyperpolarizing shift in the voltage dependence of M-current
activation. The calculated shift of 21 mV at 10 µM
retigabine was less than that (34 mV) for the shift of the KCNQ2/3
activation curve. However, the full activation curve of the native M
current in SCG neurons could not be determined because of interference
by other currents at positive potentials, so the shift was estimated
from that component of voltage-dependent current blocked by 10 µM linopirdine. When measured from the enhancement of
outward currents at 20 mV, retigabine had a similar potency on native
M currents (EC50 0.74 µM)
(Fig. 10) as that predicted (~1 µM) from the
shift of the KCNQ2/3 activation curves, and as that reported previously
for the enhancement of KCNQ2/3 outward currents (0.34 µM) (Wickenden et al., 2000). Hence, these
results provide additional support for the view (Wang et al., 1998)
that the native ganglionic M current is carried by KCNQ channels.
Interestingly, the retigabine-enhanced current, although still
sensitive to inhibition by muscarinic receptor stimulation, was
inhibited less completely than the native current. Because the
difference (~25%) was much less than the proportionate increase in M
current at the test potential (20 mV) produced by retigabine, this
cannot be attributed to a complete loss of response of
retigabine-modified channels. Instead, it suggests that retigabine
affects the channels in such a way as to diminish the extent to which
individual M channels are inhibited by muscarinic receptor stimulation,
possibly through some form of "allosteric" effect.
From a functional viewpoint, the shift in voltage dependence of the M
current has the consequence that, in the presence of 10 µM retigabine, a substantial fraction (30-40%) of the M
current became activated at the normal resting potential (approximately 60 mV under perforated-patch recording conditions) (Selyanko et al.,
1992). Hence, retigabine induced a substantial outward current, leading
to membrane hyperpolarization. In addition, the enhanced M current
further dampened the ability of the neuron to generate action
potentials during an imposed depolarization and abbreviated the
discharge train. If replicated in central neurons, this would provide a
satisfactory mechanistic explanation for the reported anti-epileptic
action of retigabine.
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FOOTNOTES |
Received Jan. 29, 2001; revised May 11, 2001; accepted May 14, 2001.
This work was supported by grants from the UK Medical Research Council,
the Wellcome Trust, and Glaxo SmithKline, UK. We thank Dr. Mala
Shah for tissue culture, Drs. Derek Trezise and Martin Main (Glaxo
SmithKline, Stevenage, UK) for retigabine and helpful discussions, Dr.
D. McKinnon for hKCNQ2 and rKCNQ3 cDNAs, Dr. M. T. Keating for
hKCNQ1, and Dr. T. J. Jentsch for KCNQ4. We are grateful to Dr.
A. A. Selyanko for all his suggestions and discussions.
Correspondence should be addressed to Prof. D. A. Brown,
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. E-mail: d.a.brown{at}ucl.ac.uk.
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TOP
ABSTRACT
INTRODUCTION
