Block of delayed-rectifier potassium channels by reduced haloperidol and related compounds in mouse cortical neurons

Article abstract of DOI:10.1124/jpet.105.086561

Haloperidol is known as an antagonist of dopamine D2 receptors. However, it also blocks a variety of ion channels at concentrations above the therapeutic range. Reduced haloperidol (R-haloperidol), one of the main metabolites of haloperidol, has been repo

Full text of DOI:10.1124/jpet.105.086561

0
022-3565/05/3151-352–362$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics
JPET 315:352–362, 2005
Vol. 315, No. 1
86561/3052839
Printed in U.S.A.
Block of Delayed-Rectifier Potassium Channels by
Reduced Haloperidol and Related Compounds in
Mouse Cortical Neurons
Shi-Bing Yang, Felix Major, Lutz F. Tietze, and Marjan Rupnik
European Neuroscience Institute, G o¨ ttingen, Germany (S.-B.Y., M.R.); and Institut f u¨ r Organische und Biomolekulare Chemie,
Georg-August-Universit a¨ t, G o¨ ttingen, Germany (F.M., L.F.T.)
Received March 18, 2005; accepted July 7, 2005
ABSTRACT
Haloperidol is known as an antagonist of dopamine D2 recep- The binding site of R-haloperidol is on the cytoplasmic side
tors. However, it also blocks a variety of ion channels at con- of the channel because its quaternary derivative preferen-
centrations above the therapeutic range. Reduced haloperidol tially inhibited the currents from intracellular side. 4-Chloro-
(
R-haloperidol), one of the main metabolites of haloperidol, has phenyl-4-hydroxypiperidine (4C4HP) is the active fragment of
been reported to accumulate in certain tissues, particularly in haloperidol because other compounds containing this moiety,
brain cortex, and it may produce the pharmacological effects including L-741,626 (3-[4-(4-chlorophenyl)-4-hydroxypiperidin-
associated with haloperidol treatment. In this study, we as- L-yl]-methyl-1H-indole) and loperamide, also blocked KDR
sessed the effect of R-haloperidol and other related com- channels. The potency of the 4C4HP fragment positively cor-
pounds on native delayed-rectifier potassium channels (KDR) in related with the hydrophobicity index (clogP) of the compounds
mouse cortical neurons by using the whole-cell patch-clamp tested. We conclude that R-haloperidol is a KDR channel
technique. Although R-haloperidol has much lower affinity to blocker, although it does not interfere with the normal channel
D2 receptors than haloperidol, the IC50 of R-haloperidol to function at a clinically relevant concentration.
block KDR currents was 4.4 ␮M, similar to its parent compound.
Haloperidol is commonly prescribed in clinical practice to pointes, a lethal type of cardiac arrhythmia, has been corre-
treat psychiatric and neurologic diseases and to control the lated with blocking of the HERG channel by certain com-
symptoms of Tourette’s syndrome. Its pharmacological effect pounds, including haloperidol (Brown et al., 2004; Testai et
is believed to be via the block of D2 dopamine receptors in the al., 2004). Therefore, understanding the basic mechanism of
central nervous system (Seeman and Van Tol, 1994). How- drug-channel interaction may provide useful information for
ever, effects of haloperidol other than D2 receptor antago- structure-based drug design (Fermini and Fossa, 2003).
nism have been observed. Up-regulation of A-type potassium
Potassium channels have been found in all the known
channel on chronic haloperidol treatment has been proposed organisms and exhibit a high level of heterogeneity (Jan and
to suppress neuronal activity (Hahn et al., 2003). Conversely, Jan, 1997). Aberrant activity of potassium channels has been
acute application of haloperidol has been reported to block shown in pathophysiological conditions, such as neurologic
numerous types of ion channels, including ATP-sensitive po- disorders, cardiac arrhythmia, and diabetes (Shieh et al.,
tassium channel (Yang et al., 2004), G protein-activated in- 2000). Therefore, the therapeutic and toxicologic aspects of
wardly rectifying potassium channel (Kobayashi et al., 2000), drug-induced alterations in potassium channel activity are
and human ether-a-go-go-related gene (HERG) potassium particularly relevant (Fermini and Fossa, 2003; Testai et al.,
channels (Shuba et al., 2001). Drug-induced torsade de 2004). Slow opening and lack of a fast inactivation process
characterize delayed-rectifier potassium channels (KDR). Be-
cause of the nature of its kinetics, modification of KDR chan-
Neurosciences of the International Max Planck Research School in G o¨ ttingen, nel activity affects the shape and duration of action poten-
S.-B.Y. is a Ph.D. student of the International M.D./Ph.D. Program in the
Germany.
tials (Bekkers, 2000). Prolonged opening of the channels can
Article, publication date, and citation information can be found at
lead to elevation of the local extracellular potassium concen-
http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.086561.
ϩ
tration [K ] . Under pathological conditions, even a moder-
o
ABBREVIATIONS: HERG, human ether-a-go-go-related gene; KDR, delayed-rectifier potassium channel(s); 4C4HP, 4-chlorophenyl-4-hydroxypi-
peridine; 3FBPA, 3-fluorobenzoyl propionic acid; R-haloperidol, reduced haloperidol; 4-AP, 4-aminopyridine; L-741,626, 3-[4-(4-chlorophenyl)-
4-hydroxypiperidin-L-yl]methyl-1H-indole; N-Me-R-haloperidol, N-methyl-reduced haloperidol.
3
52

R-Haloperidol Block of KDR Channels
353
ϩ
ate increase in [K ] drastically influences neuronal excit- NaOH). In certain experiments, equimolar KCl or RbCl was used to
o
ability and synaptic transmission (Moulder et al., 2004). On replace NaCl, and pH was adjusted by adding KOH or RbOH, re-
ϩ
spectively. The intracellular solution contained 150 mM KCl, 10 mM
the other hand, [K ] also regulates potassium channel ki-
o
HEPES, 1 mM MgCl , 5 mM Na ATP, and 5 mM EGTA, with pH
netics (Kuo, 1997), and interactions between drugs with ion
channels can be modified at different [K ] (Kuo, 1998; Jo et
al., 2000; Yang et al., 2004).
In humans, haloperidol is metabolized via three major
pathways. One metabolic route is oxidative N-dealkylation
by cytochrome P450 and generates two metabolites, 4-chloro-
2
2
ϩ
adjusted to 7.2 (by KOH). Na
added to block ATP-sensitive potassium currents (IKATP) and Ca
2
ATP (5 mM) and EGTA (5 mM) were
o
2ϩ
-
activated potassium currents (IKCa) (Speier et al., 2005). Contami-
nation by voltage-activated sodium and calcium currents at voltage
steps to ϩ100 mV was negligible; therefore, no blockers were added
to the extracellular solution. KCl (150 mM) in the intracellular
4
-hydroxypiperidine (4C4HP) and 3-fluorobenzoyl propionic solution was substituted with equimolar RbCl in some experiments.
acid (3FBPA). About 20% of haloperidol is degraded via this In these experiments, a minimum of 2 min was used for dialysis to
pathway. Another 50 to 60% of haloperidol is glucuronidated diminish the contamination with residual intracellular potassium
ions. The osmolarity of all the solutions was 300 Ϯ 10 mOsm/l. The
at the hydroxyl group and excreted in the urine. Alterna-
recording chamber had a volume of 2 ml, and chemicals were focally
tively, about 20 to 30% of haloperidol is reduced at the car-
applied to the cell through a self-made manifold pipe and driven by
bonyl group by ketone reductase and converted to reduced
gravity with a flow rate of 5 ml/min.
haloperidol (R-haloperidol) (Froemming et al., 1989; Kudo
L-741,626, sulpiride, and loperamide were purchased from Tocris
and Ishizaki, 1999). R-Haloperidol is believed to have little
clinical relevance because the binding affinity to D2 receptors
is fairly low compared with haloperidol (Kirch et al., 1985).
Cookson Inc. (Bristol, UK), and 4C4HP was obtained from Alfa Aesar
(
Ward Hill, MA). All the other chemicals were from Sigma-Aldrich
Laborchemikalien (Seelze, Germany). Haloperidol, R-haloperidol,
However, its prolonged half-life and bigger volume of distri- 4C4HP, 3FBPA, sulpiride, and L-741,626 were dissolved in dimethyl
bution (V ) than haloperidol produces a substantial buildup sulfoxide. The final concentrations of dimethyl sulfoxide were less
d
of R-haloperidol in certain tissues (Korpi et al., 1984). Be- than 0.1%, which did not affect the potassium currents tested.
N-Methyl-R-haloperidol (N-Me-R-haloperidol) was synthesized from
cause it can be oxidized back to haloperidol through the
haloperidol as follows. NaBH4 (50 mg, 1.33 mmol) was added to a
cytochrome P450 system, R-haloperidol has been proposed as
stirred solution of haloperidol (500 mg, 1.33 mmol) in MeOH (50 ml) at
an in vivo haloperidol reservoir (Kudo and Ishizaki, 1999).
room temperature for 80 min; water (10 ml) then was added. After 15
In this study, we investigated the effect of R-haloperidol
min, the solvent was removed in vacuo, and the resulting crude product
and other chemically related compounds on KDR channels in
mouse cortical neurons. We found that R-haloperidol is an
open channel blocker for KDR channels. The binding site of
was purified by column chromatography on silica gel (CH Cl /MeOH,
2
2
1
0:1) to provide R-haloperidol (450 mg, 90%) as a white powder.
A solution of R-haloperidol (100 mg, 265 ␮mol) in acetone (1 ml)
R-haloperidol is located at the intracellular side of the chan- was treated drop-wise with CH I (18 ␮l, 291 ␮mol) and heated under
3
nel. Binding of R-haloperidol to the K
channel is modu- microwave irradiation conditions (Personal Chemistry SmithCreator
microwave reactor; Biotage, Charlottesville, VA) at 60°C for 1 h. The
solvent was then removed in vacuo, and the resulting crude product
was recrystallized from CH Cl to give the desired N-Me-R-haloper-
DR
ϩ
lated by [K ] .
o
2
2
Materials and Methods
idol iodide (85 mg, 62%) as a pale yellow solid, which was subjected
1
13
Primary Culture of Mouse Cortical Neurons. All the animal to H NMR, C NMR, and mass spectroscopy. The obtained spectra
studies were conducted according to the National Institutes of matched the proposed structure of N-Me-R-haloperidol iodide.
Health’s Guidelines for Care and Use of Experimental Animals and
Data Analysis. Curve fitting was done using PulseFit v8.65
were approved by the Committee on Animal Care and Use of the (HEKA) and SigmaPlot v8.0 (SPSS Inc., Chicago, IL). Data are given
local institution and state. Pregnant female C57/BL6 mice were as mean Ϯ S.E.M., and n indicates the number of cells analyzed.
killed by cervical dislocation, and embryos (E15–E17) were extracted Statistical significance of the difference between the samples was
from the uterus. Brain cortices were isolated from embryos and were determined using Student’s t test. The clogP value of each compound
6
Ϫ1
dissociated by gentle trituration. Cells (5 ϫ 10 ml ) were plated was predicted by using OSIRIS software (Actelion, South San Fran-
onto glass coverslips pretreated with laminin and poly-L-ornithine cisco, CA). The pK value of R-haloperidol was predicted by
a
and cultured in chemically defined medium containing basal me- SciFinder database (Chemical Abstracts Service, Columbus, OH).
dium Eagle (Invitrogen, Carlsbad, CA) supplied with 2% B27 sup-
plement (Invitrogen), 1% glucose, and 1% fetal bovine serum (In-
vitrogen). Cortical neurons were selected based on the pyramidal
morphology and were used between 6 and 8 days in vitro.
Electrophysiology. Potassium currents were recorded from cul-
tured cortical neurons using standard whole-cell patch-clamp configu-
ration by an EPC10 amplifier (HEKA, Lambrecht/Pfalz, Germany).
Results
Separation of Voltage-Gated Potassium Currents in
Mouse Cortical Neurons. A large family of voltage-gated
potassium channels are presented in mammalian neurons
Data were acquired at 20 kHz with PULSE8.6 software (HEKA). Pi- (Trimmer and Rhodes, 2004). We used the standard pharma-
pettes were pulled from 1.5-mm borosilicate glass capillaries (Harvard cological method to isolate KDR currents by including 4-AP
Apparatus Inc., Holliston, MA), and their tips were heat-polished using into the extracellular solution. Figure 1A shows typical cur-
a microforge (MF830; Narishige, Tokyo, Japan). Pipette resistances
rents recorded from a cortical pyramidal neuron. The fast
inactivating potassium currents (A-type K currents) were
suppressed by holding cells at Ϫ40 mV and adding 4-AP to
the extracellular solutions further decreased the peak cur-
rents (Fig. 1B). The residual currents were insensitive to
were 3 to 5 M⍀ in standard intracellular solution. Recordings with
series resistance higher than 20 M⍀ were excluded from this study.
Unless otherwise indicated, the KDR currents were elicited by 400-ms
voltage steps from holding potential at Ϫ40 mV to ϩ100 mV every 6 s.
The capacitive transients were subtracted online by the P/4 method. All
the experiments were done at room temperature (20–25°C).
ϩ
4
-AP and can be further blocked by tetraethylammonium
extracellularly (data not shown), which resembles the typical
KDR currents (Bekkers, 2000). To reduce the contaminations
Solutions and Chemicals. The extracellular solution contained
1
50 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl , 1 mM
2
MgCl , and 5 mM 4-aminopyridine (4-AP) with pH adjusted to 7.2 (by of A-type potassium currents with minimum effects on KDR
2

3
54
Yang et al.
Idrug
Icontrol
1
ϭ
(1)
n
͓drug͔
ͩ
1 ϩ ͩ ͪ ͪ
IC
5
0
where Idrug and Icontrol are the current amplitudes of the last
10 ms of the pulse (from 390 to 400 ms of the ϩ100-mV
voltage step) in the presence and absence of the drug, respec-
tively, IC50 is the half-maximal inhibitory concentration of
the drug applied, and n is the slope parameter (Hill coeffi-
cient).
The metabolites of haloperidol were also examined in a
similar manner. 4C4HP (Fig. 2D) and 3FPBA (Fig. 2E), two
metabolites generated by oxidative N-dealkylation, were
much less potent than haloperidol. 3FPBA scarcely blocked
the KDR. On the other hand, R-haloperidol, a metabolite
generated by carbonyl reduction, blocked the KDR currents to
a similar degree as the parent compound (Fig. 2F). Because
R-haloperidol has higher concentrations in the human brain
than haloperidol, we focused on this metabolite for the rest of
the study.
Sidedness of R-Haloperidol Block. Potassium channels
possess multiple drug binding sites, and some are only ac-
cessible from one side of the plasma membrane. R-haloperi-
dol is a tertiary amine, and it can either directly bind to its
binding site from the extracellular side or diffuse across the
plasma membrane and dock to the binding site on the cyto-
plasmic side (Hille, 1977). To explore the sidedness of R-
haloperidol binding, we synthesized N-Me-R-haloperidol, the
quaternary amine derivative of R-haloperidol (Fig. 3A). Be-
cause it contains a permanent charge, it cannot cross the
plasma membrane easily as its tertiary amine form when
applied to either side of the membrane. Extracellular N-Me-
R-haloperidol only partially blocks the KDR current even at
concentrations above 100 ␮M (Fig. 3, B and D), but 100 ␮M
N-Me-R-haloperidol significantly blocked the KDR currents
when included in the intracellular solutions (Fig. 3, C and D).
The diffusion of intracellular N-Me-R-haloperidol was so fast
that the characteristic blocking kinetics could be observed in
the first trace after the establishment of whole-cell configu-
ration (Fig. 3C). Assuming N-Me-R-haloperidol binds to the
same binding site as R-haloperidol, these experiments im-
plied that R-haloperidol has to cross the membrane to access
its binding site.
Voltage Dependence of R-Haloperidol Block. R-halo-
peridol has a predicted pKa value of 8.5. In our standard
extracellular solution, pH 7.2, about 95% of R-haloperidol
was positively charged; thus, the membrane voltage can in-
fluence the binding of R-haloperidol if the binding site locates
Fig. 1. Pharmacological separation of potassium currents in mouse cor-
tical neurons. A, potassium currents were elicited by stepping from hold- in the electric field (Garcia-Ferreiro et al., 2004). To assess
ing potential Ϫ40 mV to various voltages between Ϫ40 and ϩ100 mV. the voltage-dependent block of R-haloperidol on KDR cur-
Different concentrations of 4-AP were added as indicated. B, peak cur-
rents, we used 400-ms voltage steps from Ϫ40 mV to poten-
rents under different control and different 4-AP concentrations were
tials between ϩ20 and ϩ100 mV (Fig. 4A). R-Haloperidol (10
plotted against the stepping voltages (n ϭ 6–11 for each point).
␮
M) blocked these currents to a similar extent (Fig. 4B). To
currents, 5 mM 4-AP was added into the extracellular solu-
tions.
quantify the voltage dependence of the R-haloperidol block,
the normalized currents were plotted against the membrane
potential and fitted by the Woodhull equation (Choi et al.,
Block of KDR Currents by Haloperidol and Its Metab-
olites. We first examined the block of KDR current by halo-
peridol and its metabolites listed in Fig. 2A. Haloperidol
blocked the KDR currents in a reversible manner (Fig. 2B).
The concentration-dependence curve was constructed by fit-
ting data to the Hill equation (Fig. 2C):
2001):
Idrug
͓R-halop͔
ϭ
(2)
Icontrol ͓͑R-halop͔ ϩ IC ͑0 mV͒ ϫ exp͑␦FV/RT͒͒
50

R-Haloperidol Block of KDR Channels
355
Fig. 2. Block of KDR currents by haloperidol and its metabolites in mouse cortical neurons. A, chemical structures of haloperidol and its major
metabolites. B, KDR currents were blocked by haloperidol in a concentration-dependent manner. They were activated by a 400-ms voltage step to ϩ100
mV from Ϫ40 mV. This step voltage was chosen to activate the channels at a maximum rate. The haloperidol block was reversible on the removal of
haloperidol. C, concentration-dependent curve of haloperidol block of KDR currents. The average current amplitude measured during the last 10 ms
of the 400-ms voltage step at different haloperidol concentration was normalized to the currents in the control condition. The line was fitted by eq. 1
with IC50 ϭ 4.4 ␮M and n ϭ 0.9 (n ϭ 5). The block of major haloperidol metabolites is shown in (D) 4C4HP, (E) 3FBPA, and (F) R-haloperidol. G,
concentration-dependence curves of these metabolites. The IC50 values are 661, 4033, and 4.4 ␮M, and the Hill coefficients are 0.9, 0.9, and 0.7 for
4C4HP, 3FBPA, and R-haloperidol, respectively. The number of cells tested at each drug concentration was between four and eight.
where [R-halop] is the concentration of R-haloperidol used distance was 0.07 (Fig. 4C), indicating a weak voltage depen-
10 ␮M), IC50(0 mV) is the half-inhibition concentration of dence of R-haloperidol block.
(
؉
R-haloperidol at 0 mV, and ␦ is the fractional electrical dis-
Elevation of [K ] Reduces the Affinity of R-Haloper-
o
tance from the external surface of the pore. F is the Faraday idol to KDR Channels. Binding of haloperidol and other
constant, V is the membrane potential, T is the absolute compounds to the potassium channels is sensitive to the
ϩ
temperature, and R is the gas constant. A value of 25 was [K ] (Kuo, 1998; Boccaccio et al., 2004; Yang et al., 2004).
o
ϩ
used for RT/F because the experiments were performed at The interaction of R-haloperidol and [K ] was tested by
o
ϩ
room temperature (20°C). The calculated fractional electrical increasing the [K ] from 5 to 150 mM, and the degree of
o

3
56
Yang et al.
Fig. 3. Sidedness of N-Me-R-haloperidol block on KDR currents. A, the
chemical structure of N-Me-R-haloperidol. B, 100 ␮M N-Me-R-haloperi-
dol was applied to the extracellular solution, and it only partially inhib-
ited the KDR currents. C, when 100 ␮M N-Me-R-haloperidol was included
into the intracellular solution, it gradually blocked the KDR currents on
diffusion. First trace after the membrane rupture and a representative
trace reaching steady state were shown here. D, comparison of 100 ␮M
N-Me-R-haloperidol block from different sides of the membrane. KDR
currents were significantly smaller in the presence of intracellular N-Me-
R-haloperidol (n ϭ 8 and 6 for extracellular and intracellular N-Me-R-
haloperidol, respectively; p Ͻ 0.05).
Fig. 4. Voltage dependence of R-haloperidol block of KDR currents. Cur-
rents were obtained by applying 400-ms voltage steps from Ϫ40 mV to
voltages between ϩ20 and ϩ100 mV with 20-mV increments every 6 s.
The representative traces under control conditions are shown in (A), and
the traces after adding 10 ␮M R-haloperidol are shown in (B). The initial
inward currents are sodium and calcium, which are also partially blocked
by 10 ␮M R-haloperidol. C, the fraction of block by 10 ␮M R-haloperidol
was plotted against the test voltages. The line is drawn by eq. 2, which
R-haloperidol block of K
[
currents was decreased at high yielded ␦ ϭ 0.07 and IC50(0 mV) ϭ 4.0 ␮M (n ϭ 8–9 at each voltage).
K ] (Fig. 5, A and B). The concentration-dependence curve
DR
ϩ
o
was shifted toward the right, and the IC50 of R-haloperidol
ϩ
concentration of R-haloperidol (Fig. 6, E and F). This result
suggests a bimolecular interaction between R-haloperidol
and channels in the open state as shown in the following
scheme:
was elevated from 4.4 ␮M at 5 mM [K ] to 21 ␮M at 150 mM
o
ϩ
[
K ] (Fig. 5E). However, when potassium ions on both sides
o
of the membrane were substituted by rubidium, another
ϩ
potassium channel permeant ion, the Rb currents were
blocked by R-haloperidol to a similar extent, irrespective of
koff
ϩ
[
Rb ] (Fig. 5, C, D, and F).
o
C 3 O -|0 OD
R-Haloperidol not only decreased the current amplitude
but also changed the kinetics by accelerating the current
kon
decay (Fig. 2F). This implied that R-haloperidol preferen- where C and O represent the open and closed state of chan-
tially binds to an open state of the channels. We further nels, and D denotes the R-haloperidol.
analyzed the kinetics of the R-haloperidol binding and the
effect of external ions on the binding kinetics. Current traces
at different R-haloperidol concentrations were point-by-point
divided by the trace recorded in control conditions, and these
normalized traces were fitted by a single exponential func-
tion (Fig. 6, A–D):
The block rate can be expressed as:
␶Ϫ1 ϭ kon ϫ ͓R-Halop͔ ϩ koff
(4)
kon and koff stand for the on-rate and off-rate of R-haloperidol
to the potassium channels, and [R-halop] is the concentration
of R-haloperidol.
The k values were comparable at either 5 or 150 mM
Idrug
Icontrol
Ϫt
␶
on
ϩ
o off o
ϩ
ϭ a ϩ b expͩ ͪ
(3) [K ] , but the k values were accelerated at 150 mM [K ]
(
Fig. 6E). In contrast, the binding and unbinding kinetics of
ϩ
Ϫ1
The block rate (␶ ) is the inverted decay time constant of R-haloperidol were insensitive to [Rb ] (Fig. 6F); the kon
o
each normalized current trace and was increased at higher was about twice as fast in rubidium solutions than in potas-

R-Haloperidol Block of KDR Channels
357
Fig. 5. Effect of permeant ions on the R-haloperidol block of KDR channels. Currents were elicited by a 400-ms voltage step from Ϫ40 mV to ϩ100 mV.
ϩ
ϩ
Representative traces of potassium currents blocked by R-haloperidol at (A) 5 mM [K ] and (B) 150 mM [K ] . When rubidium ions were used as
o
o
ϩ
ϩ
charge carriers, the degree of R-haloperidol block was comparable at both (C) 2 mM [Rb ] and (D) 150 mM [Rb ] . E, concentration-dependence
o
o
ϩ
inhibition curve of R-haloperidol at different [K ] . The last 10 ms of current traces at different R-haloperidol concentration were normalized to the
o
ϩ
current at control condition. The data were fitted by eq. 1. For comparison, the data points of 5 mM [K ] are taken from Fig. 2G. The IC value was
o
50
ϩ
2
1.0 ␮M with Hill coefficients of 0.7 at 150 mM [K ] . F, concentration-dependence inhibition curves of R-haloperidol block of rubidium currents at
o
ϩ ϩ ϩ
different [Rb ] . IC values were 6.3 and 5.0 ␮M, and the Hill coefficients were 0.9 and 0.7 for 2 mM [Rb ] and 150 mM [Rb ] , respectively. For each
o
50
o
o
data point in (E) and (F), 2 to 18 cells were used with a median cell number of seven.
ϩ
sium solutions, and the k at both [Rb ] was comparable state were also decreased in the presence of extracellular
off
ϩ
with the koff at low [K ] (Fig. 6A).
o
potassium ions (Fig. 6G).
o
The intercept of the normalized current traces on the y-axis
Block of KDR Channels by Compounds Functionally
represents the fraction of channels blocked before they or Structurally Related to Haloperidol. The backbone
opened (Fig. 6, A–D). Therefore, the affinity of R-haloperidol structure of R-haloperidol is similar to haloperidol, although
to the channels in the closed state can be estimated by the affinity to D2 receptor is reduced by about two orders of
plotting the y-intercept versus R-haloperidol concentration. magnitude (Kirch et al., 1985; Froemming et al., 1989). Thus,
The apparent binding affinities to the channel in the closed binding of R-haloperidol to KDR channels does not correlate

3
58
Yang et al.
Fig. 6. Kinetics of R-haloperidol block at different ionic conditions. A–D, original current traces were recorded as in Fig. 4. The currents at the
indicated extracellular cation concentration were normalized point by point to the control current trace without the addition of R-haloperidol and fitted
ϩ
with eq. 3. E, calculated block rates at different [K ] were plotted against the R-haloperidol concentration. According to eqs. 4 and 5, a straight line
o
was used to fit the R-haloperidol binding kinetics. The slope and the y-intercept represent the on-rate (k ) and the off-rate (koff), respectively. The
on
ϩ
ϩ
on-rates were comparable at different [K ] , and the values were 4.9 ␮M⅐s and 5.6 ␮M⅐s for 5 and 150 mM [K ] , respectively. The k was increased
from 39/s to 66/s as the [K ] increased from 5 to 150 mM. In contrast, the R-haloperidol binding kinetics were almost indistinguishable in different
o
o
off
ϩ
o

R-Haloperidol Block of KDR Channels
359
Fig. 7. KDR currents blocked by other compounds with the 4C4HP moiety. A, chemical structures of compounds tested. B, representative current traces
in the presence and absence of sulpiride. Only 2 Ϯ 2% of currents were blocked by 100 ␮M sulpiride (n ϭ 4). C, representative traces recorded at
different concentrations of L-741,626. D, concentration-dependence curve of L-741,626 block on KDR currents. The IC50 and Hill coefficient were 3.3
␮
M and 0.8, respectively (n ϭ 5). E, representative traces recorded at different concentrations of loperamide. F, concentration-dependence curve of
loperamide block on KDR currents. The IC50 and Hill coefficient were 0.75 ␮M and 0.6, respectively (n ϭ 5).
to the D2 receptor potency. To address the active chemical a similar extent as R-haloperidol (Fig. 7, C and D). If the
moiety, several compounds with either functional or struc- chlorophenyl piperidine moiety determines the block ability,
tural similarity were tested (Fig. 7A). Sulpiride, another D2 other compounds with this fragment should also block the
receptor antagonist with similar potency as haloperidol but channels. Loperamide, an antidiarrheal drug that activates
structurally unrelated, did not inhibit the potassium cur- opioid receptors, also contains this fragment. Therefore, we
rents when applied extracellularly (Fig. 7B). In contrast, tested whether loperamide also blocked the KDR currents. To
L-741,626, a D2 receptor antagonist with a similar chloro- avoid the activation of opioid receptor, 10 ␮M naloxone was
phenyl piperidine moiety, blocked the potassium currents to continuously present in the extracellular solution before and
ϩ
ϩ
[
Rb ] . F, the k values were 11 ␮M⅐s and 10 ␮M⅐s for 2 and 150 mM [Rb ] , respectively, which were about twice as fast as in the solutions containing
o on o
ϩ
potassium. The koff values were 32/s and 38/s for 2 and 150 mM [Rb ] , respectively. G, affinity of R-haloperidol to closed-state channels. The
o
y-intercepts in (A–D) were the portion of channels blocked before the opening, and hence the affinity to the closed-state channels was estimated by
fitting the concentration-dependence curves at various R-haloperidol concentrations. The lines were fitted by eq. 1. The IC values were 76 ␮M, 98
5
0
ϩ
ϩ
ϩ
ϩ
␮
M, 52 ␮M, and 47 ␮M for 5 mM [K ] , 150 mM [K ] , 2 mM [Rb ] , and 150 mM [Rb ] , respectively. The Hill coefficients were 1.1, 1.8, 1.0, and 1.3
o o o o
ϩ ϩ ϩ ϩ
for 5 mM [K ] , 150 mM [K ] , 2 mM [Rb ] , and 150 mM [Rb ] , respectively. The numbers of cells tested for each point were between 2 and 11
o
o
o
o
(
median, 5).

3
60
Yang et al.
during the application of loperamide. KDR currents were not knock off the R-haloperidol from its binding site. According to
affected in the presence of 10 ␮M naloxone (data not shown). the structure of a potassium channel, the selective filter has
We found that loperamide was even more potent than other different conformation when crystallized in solutions with
ϩ
ϩ
compounds tested, with an IC50 value of 0.75 ␮M (Fig. 7, E different K concentrations, but not Rb . Based on this sce-
ϩ
and F).
nario, the selective filter has a low energy barrier for K , and
this ion can freely move across the filter, but it functions aϩs
a molecular hurdle for other permeant ions, for example, Rb
Discussion
ϩ
or Cs ; even these ions can traverse through the channel as
K (Morais-Cabral et al., 2001; Zhou and MacKinnon, 2003).
ϩ
In this study, we have shown that R-haloperidol, a metab-
ϩ ϩ
olite of haloperidol, is an open channel blocker for KDR chan- Increase of external K but not Rb boosts the ion flow
nels in mouse cortical neurons. R-Haloperidol accelerates the through the permeant pathway and therefore pushes R-hal-
current decay in a concentration-dependent manner. Elevat- operidol out from the binding site. Recently, Lin et al. (2005)
ϩ
ϩ
ϩ
ing the [K ] antagonized the binding of R-haloperidol to K
reported a similar phenomenon that K but not Cs regu-
o
DR
channels. The binding site is shown to be at the intracellular lates the binding of cisapride, a prokinetic compound, to
side. The 4C4HP fragment determines the KDR channel block HERG channels. Nevertheless, the possibility of allosteric
ϩ
ability, although the fragment itself only weakly blocks the modulation of a channel by K cannot be completely ruled
channels.
out.
Nature of the Binding Motif on R-Haloperidol. The
Binding Site of R-Haloperidol on the KDR Channels.
According to the scheme shown in eq. 4, R-haloperidol binds ability of compounds to block the KDR channels is dependent
to the KDR channel in the open state. If the binding of R- on the existence of the 4C4HP fragment but is not related to
haloperidol to the KDR channels did not have the preferential the pharmacological character of the compounds. We found a
state, a proportional reduction of current size should have linear relationship between the logIC50 and calculated the
been expected without altering the channel kinetics. There hydrophobicity index (clogP) in compounds containing the
are two lines of evidence suggesting that R-haloperidol has a 4C4HP fragment (Fig. 8). We proposed that the 4C4HP is
higher affinity to open KDR channels. First, the current de- the key fragment that binds to the KDR channel, and the
cayed faster at higher R-haloperidol concentration (Fig. 6). affinity of 4C4HP is enhanced by adding a hydrophobic motif
Equation 4 predicts a one-to-one relationship between R- at the nitrogen atom on the piperidine ring, which increases
haloperidol and the KDR channel, and the binding rate the accessibility of 4C4HP to its intracellular binding site. On
should be linearly related to the ligand concentration. Sec- the other hand, increase of hydrophobicity also facilitates the
ond, the IC50 of R-haloperidol to the closed channels was binding of the active fragment to the receptor, and the hy-
estimated to be 76 ␮M, which was more than 10 times higher drophobic motif functioned as a “binder” of the active frag-
than 4.4 ␮M, the IC50 of the R-haloperidol block to the open ment. The binder concept was recently suggested in a frag-
channels (Figs. 2G and 5G). The voltage gate of potassium ment-based lead discovery, in which optimizing the binder
channels is located at the cytoplasmic end of the channel part of the molecule augments the binding affinity (Rees et
protein (Jiang et al., 2002). Therefore, we propose that the al., 2004). It has been shown that increase of the hydropho-
binding site of R-haloperidol is either formed or accessible bicity of quaternary amines enhances the binding affinity to
when the channel is opened. The quaternary amine binding its binding site (Choi et al., 1993). The chemical nature of the
has been located in the central cavity of the channel (Zhou et binder can be unspecific because the key fragment deter-
al., 2001), and we also have shown that the N-Me-R-haloper- mines the main biological effect. Therefore, it is worth check-
idol, the quaternary amine derivative of R-haloperidol, pref- ing the channel blocking ability of compounds with the
erentially blocked the KDR currents from the intracellular 4C4HP motif at an early stage of drug development to avoid
side (Fig. 3). The fractional electrical distance of 0.07 indi- adverse effects caused by ion channel blocking (Fermini and
cated a weak voltage dependence of R-haloperidol block (Fig. Fossa, 2003).
4
). When the channel is in its open configuration, the voltage
decreases significantly across the selective filter, and the
central cavity is at the same membrane potential as the
cytosolic solution (Jiang et al., 2002). If the R-haloperidol
binding site is in the central cavity and it is only accessible
when the channel is open, it is plausible that the R-haloper-
idol block is independent of membrane voltage. Nevertheless,
we cannot exclude the possibility that R-haloperidol blocks
the channel in its neutral form.
Interaction of R-Haloperidol and External Cations
ϩ
on the KDR Channels. Raising of [K ] reduced the R-
o
haloperidol binding affinity to its binding site, whereas an
ϩ
increase in [Rb ] , another potassium channel permeant ion,
o
does not produce a similar effect (Figs. 5 and 6). Interactions
between permeant ions and blockers have been observed
previously (Kuo, 1998; Boccaccio et al., 2004; Yang et al.,
ϩ
Fig. 8. Relationship between the potency and hydrophobicity of com-
pounds studied. The binding affinities of each compound were plotted
against the clogP values. A straight line with a slope of 1.0 with a
2
004; Yang and Kuo, 2005). The main effect of external K is
accelerating the unbinding of R-haloperidol from the K
channel; therefore, it seems that only K , but not Rb , can y-intercept of Ϫ0.8 was used to describe the relationship.
DR
ϩ
ϩ

R-Haloperidol Block of KDR Channels
361
The oxygen atom in the carbonyl group has been proposed
to involve the binding of haloperidol to the central cavity of
HERG channels in an in silico study (Testai et al., 2004).
Most compounds listed in that study have already been re-
ported as blockers for other potassium channels, including
KDR channels. R-Haloperidol, in contrast, has a hydroxyl
group at this position. Nevertheless, the binding affinities to
the KDR channel of both R-haloperidol and haloperidol were
comparable. It is likely the role of carbonyl oxygen was over-
estimated in the above-mentioned in silico study (Testai et
al., 2004).
Acknowledgments
We thank A. Bohl, M. Niebeling, and H. R o¨ hse for excellent tech-
nical assistance. F.M. thanks the Studienstiftung des deutschen
Volkes for a Ph.D. scholarship.
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Address correspondence to: Shi-Bing Yang, Waldweg 33, 37073 G o¨ ttingen,
Germany. E-mail: syang1@gwdg.de