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The Journal of Neuroscience, January 15, 2002, 22(2):396-403
Differential Inhibition of T-Type Calcium Channels by
Neuroleptics
Celia M.
Santi1,
Francisco S.
Cayabyab2,
Kathy G.
Sutton1,
John E.
McRory1,
Janette
Mezeyova2,
Kevin S.
Hamming1,
David
Parker2,
Anthony
Stea3, and
Terrance P.
Snutch1
1 Biotechnology Laboratory, University of British
Columbia, Vancouver, British Columbia, Canada, V6T 1Z3,
2 NeuroMed Technologies, Vancouver, British Columbia,
Canada, V6T 1Z4, and 3 University-College of the Fraser
Valley, Abbotsford, British Columbia, Canada, V2S 7M8
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ABSTRACT |
T-type calcium channels play critical roles in cellular
excitability and have been implicated in the pathogenesis of a variety of neurological disorders including epilepsy. Although there have been
reports that certain neuroleptics that primarily target D2 dopamine receptors and are used to treat psychoses may also interact with T-type Ca channels, there has been no systematic examination of
this phenomenon. In the present paper we provide a detailed analysis of
the effects of several widely used neuroleptic agents on a family of
exogenously expressed neuronal T-type Ca channels ( 1G, 1H, and 1I
subtypes). Among the neuroleptics tested, the diphenylbutylpiperidines
pimozide and penfluridol were the most potent T-type channel blockers
with Kd values (~30-50 nM and
~70-100 nM, respectively), in the range of their
antagonism of the D2 dopamine receptor. In contrast, the
butyrophenone haloperidol was ~12- to 20-fold less potent at blocking
the various T-type Ca channels. The diphenyldiperazine flunarizine was
also less potent compared with the diphenylbutylpiperadines and
preferentially blocked 1G and 1I T-type
channels compared with 1H. The various neuroleptics did
not significantly affect T-type channel activation or kinetic
properties, although they shifted steady-state inactivation profiles to
more negative values, indicating that these agents preferentially bind
to channel inactivated states. Overall, our findings indicate that
T-type Ca channels are potently blocked by a subset of neuroleptic
agents and suggest that the action of these drugs on T-type Ca channels
may significantly contribute to their therapeutic efficacy.
Key words:
T-type; neuroleptics; cDNA; calcium channels; schizophrenia
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INTRODUCTION |
The rapid entry of calcium into
cells through voltage-dependent Ca channels triggers a variety of
intracellular events such as muscle contraction, hormone secretion,
synaptic transmission, and gene expression. Ca channels have been
traditionally classified into high voltage-activated (HVA) and low
voltage-activated (LVA) subtypes (Tsien et al., 1988 ; Bean, 1989). HVA
Ca channels first activate at relatively depolarized potentials and
comprise L-, P/Q-, N-, and R-types. These channels exhibit a high
single-channel conductance and varied patterns of inactivation and
deactivation. LVA Ca channels, also known as T-type, show a more
negative range of activation and inactivation, rapid inactivation, slow
deactivation, and smaller single-channel conductances. T-type Ca
channels are present in a variety of cell types where they appear to
mediate low-threshold spikes and rebound burst firing patterns (for
review, see Huguenard, 1996), pacemaker activity (Hagiwara et al.,
1988), hormone secretion (Rossier et al., 1996), cell growth and
proliferation (Xu and Best, 1990), and are also involved in
fertilization (Arnoult et al., 1996; Santi et al., 1996). Abnormal
activity of T-type Ca channels has been implicated in the
pathophysiology of epilepsy (Huguenard and Prince, 1994; Tsakiridou et
al., 1995; Huguenard, 1996), hypertension (Self et al., 1994), and
cardiac hypertrophy (Nuss and Houser, 1993). Furthermore, the
activity of T-type channels may play a role in enlarging the area of
damage produced by a transient ischemic insult (Ito et al., 1994).
Three different T-type Ca channels have been cloned and expressed from
mammals: 1G, 1H, and
1I (Cribbs et al., 1998; Perez-Reyes et al.,
1998; Klugbauer et al., 1999; Lee et al., 1999; McRory et al., 2001).
Although HVA Ca channels have a well defined pharmacology, it has
proven difficult to identify specific high-affinity blockers of LVA Ca
channels. Ni2+ and amiloride are often
described as effective blockers of T-type channels in many native
cells, although the effects of these agents is quite variable in
different cell types (Huguenard, 1996; Todorovic and Lingle, 1998).
Neuroleptic agents comprise several chemically distinct classes of
compounds (diphenylbutylpiperidines, butyrophenones, and phenothiazine)
that act as antagonists of the D2 dopamine
receptors and are widely prescribed to treat a variety of psychiatric
disorders (Seeman et al., 1976). Interestingly, many of these drugs
also display Ca channel blocking activity, although their selectivity for the different Ca channel subtypes has not been systematically examined. Although some neuroleptics inhibit to some extent P-, N-, and
L-type Ca channels, they appear to block T-type channels with
relatively higher affinity than HVA Ca channels (Enyeart et al., 1990,
1993; Takahashi and Akaike, 1991; Sah and Bean, 1993). The
diphenylbutylpiperidines (DPBPs) pimozide and penfluridol have been
shown to block T-type currents in various cell types: heart (Enyeart et
al., 1990), adrenal fasciculata (Enyeart et al., 1993), mouse
spermatogenic (Arnoult et al., 1998), neural crest cells, and human C
cell lines (Enyeart et al., 1992). Moreover, the neuroleptic-like agent
flunarizine has also been reported to act both as a
D2 dopamine receptor antagonist and as blocker of
Ca currents in the CNS where it acts as a potent organic blocker of the
T-type currents from dissociated hypothalamic neurons (Akaike et al.,
1989) as well as hypothalamic T-type currents expressed in
Xenopus oocytes (Dzura et al., 1996).
In the present paper we explored the inhibitory effects of the
neuroleptic agents pimozide, penfluridol, haloperidol, and flunarizine
on three exogenously expressed neuronal T-type Ca channels. The results
demonstrate that the DPBP class of neuroleptics are high-affinity
T-type Ca channel blockers and suggest that Ca channel block may
represent a significant contribution to the clinical efficacy of these agents.
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MATERIALS AND METHODS |
Cell culture. Stable cell lines expressing
1G, 1H, or
1I were generated by transfecting either
1G, 1H, or
1I cDNAs (all in pcDNA3.1 vector) into human
embryonic kidney (HEK) tsa 201 cells using standard Ca-phosphate
precipitation, and clones were selected with zeocin. In some cases
standard Ca-phosphate precipitation was also used for transient
cotransfection in HEK tsa 201 cells of 1I (3 µg in pcDNA3.1 vector), CD8 (2 µg) marker plasmid, and 15 µg of
pBluescript SK carrier DNA for a total of 20 µg of cDNA mix.
Transiently transfected cells were selected for expression of CD8 by
adherence of Dynabeads (Dynal, Great Neck, NY). The cells were
grown in standard DMEM (10% fetal bovine serum and 50 U/ml
penicillin-streptomycin), maintained at 37°C in a humidified atmosphere of 95% O2 and 5%
CO2. Stably expressing cell lines were
enzymatically dissociated with trypsin-EDTA and plated on 35 mm Petri
dishes 12 hr before recordings. Functional transient expression of
1I was evaluated 24 hr after transfection.
Electrophysiological recordings. Macroscopic currents were
recorded using the whole-cell patch-clamp technique (Hamill et al.,
1981). The external recording solution contained in
mM: 2 CaCl2 or 2 BaCl2, 1 MgCl2, 10 HEPES,
40 TEA Cl, 92 CsCl, and 10 glucose, pH 7.2. The internal pipette
solution contained in mM: 105 CsCl, 25 TEA Cl, 1 CaCl2, 11 EGTA, and 10 HEPES, pH 7.2. 1G and 1I currents
were recorded in the presence of 2 mM external Ca. Because 1H Ca currents are significantly
smaller than Ba currents (McRory et al., 2001), we used Ba as a charge
carrier for 1H (Ca was also used where
indicated). Whole-cell currents were recorded using an Axopatch 200B or
200A amplifier (Axon Instruments, Foster City, CA), controlled and
monitored with a personal computer running pClamp software version 6.03 (Axon Instruments). Patch pipettes (borosilicate glass BF150-86-10;
Sutter Instruments, Novato, CA), were pulled using a Sutter
P-87 puller and fire-polished using a Narishige (Tokyo, Japan)
microforge, with typical resistances of 2.5-4 M (filled with
internal solution). Series resistance was electronically compensated by
at least 60%. Whole-cell currents that exceeded 2 nA were not
examined, minimizing voltage error (<2-3 mV). Only cells exhibiting
adequate voltage control [judged by smoothly rising current-voltage
(I-V) relationship and monoexponential decay of
capacitive currents] were included in the analysis. The bath was
connected to ground via a 3 M KCl Agar bridge.
All recordings were performed at room temperature (20-24°C).
Drugs effects were investigated using 150 msec steps to peak potentials
every 15-30 sec from a holding potential of  100 mV. Current-voltage
relations were measured by a series of 150-msec-long depolarizing
pulses applied from a holding potential of 100 mV to membrane
potential between 90 and +20 mV every 15 sec.
Data were low-pass filtered at 2 kHz using the built-in Bessel filter
of the amplifier, and in most cases, subtraction of capacitance and
leakage current was performed on-line using P/4 protocol. Recordings
were analyzed using Clampfit 6.03 (Axon Instruments), and figures were
generated using the software program Microcal Origin (version 3.78).
Data from drug concentration-response studies were fitted with the
equation y = [(A1
A2)/{1 + (x/xo)P} + A2, where
A1 is initial (= 0) and
A2 final value,
xo is IC50 (concentration causing 50% inhibition of currents), and p
gives a measure of steepness of curve.
Time courses of channel blockade were well described by single
exponential curves from which  on and the
fraction of unblocked channels at equilibrium (a = Idrug/Icontrol)
were obtained. Kd values were
estimated using the following equation:
The steady-state inactivation curves were constructed by
plotting the normalized current during the test pulse as a function of
the conditioning potential. The data were fitted with a Boltzmann equation: I/Imax = {1 + exp[V V0.5i/ki]} 1,
where I is the peak current,
Imax is the peak current when the conditioning pulse was 120 mV, V and
V0.5i are the conditioning potential
and the half-inactivation potential respectively, and ki is the inactivation slope factor.
Current-voltage relationships were fitted with the Boltzmann equation:
I = {1/1 + exp(Vm V0.5a)/ka)}{(V Erev)Gmax},
where Vm is the test potential,
V0.5a is the half-activation
potential, Erev is the extrapolated
reversal potential, Gmax is the
maximum slope conductance, and ka
reflects the slope of the activation curve.
Statistical significance was determined by Student's t test
or one-way ANOVA followed by Bonferroni multiple comparison post-test, as appropriate, and significant values were set as indicated in the
text and figure legends.
Solutions and drugs. Penfluridol was from Research
Diagnostics Inc. (Flanders, NJ). All other drugs were purchased
from Sigma (St. Louis, MO), unless otherwise specified. Test solutions
containing drugs were prepared fresh for each experiment from
concentrated stock solutions and added to the recording solution.
Concentrated stock solutions were as follows: pimozide (1 mM in DMSO); penfluridol (500 µM in ethanol), haloperidol (1 mM in DMSO), and flunarizine (1 mM in DMSO). The highest concentration of ethanol
and DMSO in the recording solution did not exceed 0.1%, a
concentration that did not detectably affect calcium channel
properties. The perfusion system consisted of a custom-made multiple
solution perfusion manifold consisting of three input and three output capillary tubes (custom microfil, 28 gauge, 250 µm inner diameter and
350 µm outer diameter; World Precision Instruments, Sarasota, FL)
ensheathed in a glass pipette. The lengths of lines from valve to the
manifold were kept to a minimum, and the outputs of the manifold were
placed within five cell lengths, resulting in cells being bathed with
new solutions with minimal delay (within 1 sec) and minimal dead space volume.
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RESULTS |
Diphenylbutylpiperidines are high-affinity antagonists of T-type
Ca channels
The DPBPs (e.g., pimozide, penfluridol) (Fig.
1A,B) are a class of
neuroleptic drugs clinically used to relieve the symptoms of
schizophrenia (Pinder et al., 1976). The DPBPs were initially identified as potential Ca channel antagonists when it was discovered that these agents inhibited dihydropyridine (DHP) binding to specific brain receptors and also inhibited depolarization-dependent Ca uptake
and contraction in muscle (Gould et al., 1983). A number of reports
have shown that DPBPs are relatively potent blockers of L-type Ca
channels in endocrine (e.g., pituitary) and heart muscle cells (Enyeart
et al., 1990). In some of these same preparations it has been observed
that low concentrations of DPBPs also block T-type currents (Enyeart et
al., 1990, 1992). While some DPBPs (e.g., pimozide) have been used to
probe the physiological functions of T-type channels (Arnoult et al.,
1998), it has not been established whether these agents are selective
for particular subtypes of T-type channel. We tested the effect of two
DPBPs, penfluridol and pimozide (Fig. 1A,B), on
exogenously expressed 1G,
1H, and 1I T-type Ca
channels from rat brain (Fig. 2).
Penfluridol and pimozide were not subtype-selective because both of
these agents blocked the three neuronal T-type Ca channels to similar
extents (Fig. 2, Table 1). A detailed
analysis of the concentration-response relations of pimozide and
penfluridol with the three T-type Ca channels revealed a similar range
of IC50 values in the case of pimozide (34.6, 53.5, and 30.4 nM for
1G, 1H, and
1I, respectively) and
IC50 values equal to 93.1, 64.1, and 71.6 nM for 1G,
1H, and 1I,
respectively, for penfluridol (Fig. 2A,B)
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Figure 1.
Chemical structures of the three different
structural classes of neuroleptic agents used in this study: the DPBPs
pimozide (A) and penfluridol
(B). An example of a butyrophenone
(C, haloperidol) and a diphenyldiperazine, flunarizine
(D).
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Figure 2.
Summary of the effect of DPBPs on T-type calcium
channels. A, B, Concentration-response curves for
pimozide and penfluridol of 1G,
1H, and 1I T-type channels. Data points
reflect mean ± SE of two to six determinations. Half-maximal
inhibition (IC50) were determined from fitting the data as
described in the methods. IC50 values for pimozide were
34.6, 53.5, and 30.4 nM, for 1G,
1H, and 1I, respectively.
IC50 values for penfluridol were 93.1, 64.1, and 71.6 nM, for 1G, 1H, and
1I, respectively. Inset, Representative
current traces in control (filled circles) and 5 min after addition of drugs (open circles). C,
D, Time course of block of 1G currents in eight
different cells exposed to 10, 50, 500, and 1000 nM
pimozide and 50, 200, 1000, and 10,000 nM penfluridol. Ca
currents were evoked every 15 sec with 150 msec step depolarizations
from 100 to 40 mV. Solid lines, Fits of single
exponential decays to a plateau. Insets, Dependence of
1/on on [drug]. Values are mean and SE of three
to five determinations. Solid line fit was made with the following
equation: 1/on = kon[drug] + koff, where
kon = 0.00432/sec,
koff = 0.000088/sec, and
Kd = 48.4 nM for pimozide,
and kon = 0.00117/sec,
koff = 0.0000087/sec, and
Kd = 135.1 nM for
penfluridol.
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Examination of current-voltage relations and kinetic parameters showed
that the DPBPs did not significantly affect these properties of T-type
Ca channels (data not shown).
The blocking rate of penfluridol was ~10 times slower than pimozide
with on values at 1 µM of
98.5 ± 17.2 sec (n = 5) for penfluridol and
10.01 ± 1.8 sec (n = 3) for pimozide (Fig.
2C,D, see for the 1G data). The
blocking effect of both drugs was faster at higher concentrations. The
time course of onset for pimozide and penfluridol block for
1G could be fitted well with a single exponential (Fig. 2C,D), and the plot of
1/on exhibited a linear concentration
dependence as would be expected for a 1:1 interaction (Fig. 2C,D,
insets). The values of the binding
(kon) and unbinding (koff) rate constants were obtained
from the slope and y-intercept, respectively, of the linear
equation with Kd values of 48.8 and 135.1 nM for pimozide and penfluridol,
respectively, which are very close to the IC50
values obtained from the dose-response curves. Washout of pimozide was
faster and more complete than for penfluridol (see Fig.
5A,B), and washout of penfluridol was largely incomplete.
The extent of recovery could be increased only slightly by holding at
more negative potentials (data not shown). Complete recovery of DPBP
block has been seen for T-type currents in some native cells (Enyeart
et al., 1992), whereas in other cell types DPBP block is reported to be
more persistent (Arnoult et al., 1998).
The butyrophenone haloperidol blocks T-type channels with lower
affinity than DPBPs
Haloperidol (Fig. 1C) is a potent butyrophenone
antipsychotic commonly used to treat disorders such as schizophrenia
(DiMascio, 1972). Cardiac arrhythmias have been associated with
haloperidol therapy (Hunt and Stern, 1995), although the mechanism for
this effect is unclear. It has been proposed that haloperidol could mediate cardiac effects by the block of repolarizing potassium currents, in particular HERG channels (Hunt and Stern, 1995). However, because T-type Ca channels are present in pacemaker heart cells, they may also be involved in the pathogenesis of arrhythmias. Figure 3 summarizes the effects of
application of 1 µM haloperidol on
1G, 1H, and
1I T-type Ca channels. The estimated
Kd values for haloperidol interaction
were similar among all three T-type channel subtypes (range, 1.2-1.4
µM) (Table 1). However, haloperidol is 12-to
20-fold less potent on the T-type channels compared with the DPBP
neuroleptics pimozide and penfluridol (Table 1). The block of
haloperidol developed faster than the other neuroleptic drugs with
on values ranging from 13 to 20 sec, and drug
washout was similarly faster and more complete (see Fig.
5C).
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Figure 3.
Summary of the effect of the butyrophenone
haloperidol, on 1G, 1H, and
1I T-type Ca channels. Mean I-V
relationships of T-type currents in the absence (filled
circles) or presence (open circles) of
haloperidol (1 µM) on currents through 1G
(A), 1H (B),
and 1I (C). Normalized mean
I-V data represent the mean ± SE from three to
eight cells. Insets show representative current traces
in absence (control, filled circles) and presence
(open circles) of 1 µM haloperidol,
elicited with test pulses to 40 mV (1G,
1I) or 30 mV (1H).
D, All currents were inhibited to similar levels
( 50% inhibition) by 1 µM haloperidol, as summarized
in this bar chart (n = 5 for
1G; n = 8 for
1H; n = 5 for
1I).
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Flunarizine exhibits subtype-specific blocking activity for T-type
calcium channels
Flunarizine is a diphenyldiperazine derivative (Fig.
1D) that has been used clinically to treat a number
of diseases such as vertigo (Olesen, 1988), migraine (Spierings, 1988),
and some heart disorders (Koch et al., 1990). Flunarizine is also a
potent anticonvulsant (Greenberg, 1987) and displays some
neuroprotective properties (Desphande and Wieloch, 1986; Rich and
Hollowell, 1990; Kaminski Schierle et al., 1999). Flunarizine has been
shown to block native L-, N-, and T-type Ca channels (Tytgat et al.,
1988, 1991, 1996), and some neurons possess T-type currents
characterized by their high sensitivity to this neuroleptic compound
(Kaneda and Akaike, 1989; Panchenko et al., 1993; Takahashi and Akaike, 1991). In certain preparations, flunarizine has been described as the
most potent organic blocker of hypothalamic T-type Ca channels (Akaike
et al., 1989; Dzura et al., 1996).
Figure 4 summarizes the effects of 1 µM flunarizine on 1G,
1H, and 1I T-type Ca
channels. Unlike the other neuroleptics studied, flunarizine displayed
a preferential block of 1G and 1I (Kd = 0.53 and 0.84 µM, respectively) (Table 1) compared with 1H (Kd = 3.6 µM) (Table 1). As with pimozide,
penfluridol, and haloperidol, flunarizine did not significantly alter
the current-voltage relations or current kinetics of the three T-type
channels (Fig. 4). The onset of blockade by flunarizine was the slowest
(Fig. 5D) of the neuroleptics
tested with on values at 1 µM ranging from 72 to 197 sec for
1G and 1I. Washout of
flunarizine was likewise slow and essentially complete.
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Figure 4.
Differential blockade of T-type Ca channels by the
diphenyldiperazine flunarizine. Mean I-V relationships
of T-type currents in the absence (filled
circles) or presence (open circles) of
flunarizine (1 µM), were obtained from normalized
currents (mean ± SE from 3-6 cells) through 1G
(A), 1H (B),
and 1I (C). Normalized mean
I-V data represent the mean ± SE from three to
six cells. Insets show representative current traces in
absence (control, filled circles) and presence
(open circles) of 1 µM flunarizine,
elicited with test pulses to 40 mV (1G,
1I) or 30 mV (1H).
D, Flunarizine (1 µM) differentially
inhibited the cloned T-type currents, inhibiting 1G and
1I more potently (~70%) than 1H
(~30%).
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Figure 5.
Time course of neuroleptic blockade of the three
T-type Ca channels. Shown are representative time courses of the
inhibitory effects of 500 nM pimozide
(A), 1 µM penfluridol
(B), 1 µM haloperidol
(C), and 1 µM flunarizine
(D) on cloned rat 1G,
1H, and 1I channels. Drug
application (first arrow) followed by washout of
drugs (second arrow) appear as indicated. The DPBPs
penfluridol (1 µM) and pimozide (500 nM),
showed on of inhibition ranging from 60.3 ± 16 to
109.9 ± 29 and from 29.5 ± 0.4 to 84.2 ± 12.4 sec
(n = 3-6) for penfluridol and pimozide,
respectively. In contrast, the butyrophenone haloperidol showed
on values from 13.3 ± 0.9 to 20 ± 2.6 sec
(n = 4). Flunarizine exhibits the slowest on-rate
(from 39.2 ± 8.8 to 197.3 ± 23.8 sec; n = 3). The T-type Ca currents were elicited by test pulses to 40 mV
(1G, 1I) or 30 mV
(1H) from 100 mV holding potential (every 15 sec before and during drug application).
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Neuroleptics affect T-type Ca channel steady-state
inactivation properties
To further characterize the biophysical effects of neuroleptics on
the three types of neuronal T-type Ca channels, a detailed analysis of
voltage-dependent inactivation of the 1G Ca
channel was performed. A conditioning pulse of 15 sec duration at
various potentials between 120 and 50 mV was applied and followed
by a test pulse to 30 mV of 150 msec duration. Figure
6 shows that addition of flunarizine,
haloperidol, pimozide, and penfluridol caused a significant
hyperpolarizing shift (from 7 to 10 mV) of V0.5inact compared with the
steady-state inactivation profiles of the 1G
currents under control conditions. Similar voltage-dependent block of
1H and 1I channels by
penfluridol, haloperidol, or flunarizine is suggested by comparing data
at more depolarized potentials (i.e., 85 vs 100 mV; data not
shown).
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Figure 6.
Neuroleptic agents induce a negative shift in the
voltage dependence of steady-state inactivation of 1G
T-type Ca channels. The V0.5i values of rat
1G channel inactivation were shifted to hyperpolarized
potentials as follows: A, 50 nM pimozide
(control V0.5i = 78.6 ± 0.2 mV
vs treated V0.5i = 85.8 ± 0.2 mV; p < 0.01; n = 4).
B, 100 nM penfluridol (control
V0.5i = 78.5 ± 0.3 mV vs
treated V0.5i = 90.2 ± 0.3 mV;
p < 0.01; n = 4).
C, 1 µM haloperidol (control
V0.5i = 80.8 ± 0.2 mV vs
treated V0.5i = 91.1 ± 0.3 mV;
p < 0.01; n = 3).
D, 100 nM flunarizine (control
V0.5i = 81.6 ± 0.3 mV vs
treated V0.5i = 90.4 ± 0.2 mV;
p < 0.01; n = 4). Steady-state
inactivation curves were generated as described in Materials and
Methods.
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Whereas inactivation-gating processes were profoundly affected by all
neuroleptic drugs tested, the voltage dependence of activation was not
significantly altered. The V0.5 of
activation obtained from I-V curves were not significantly
shifted (<5 mV) in the presence of the drugs (data not shown).
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DISCUSSION |
Neuroleptics represent chemically diverse classes of compounds
that share the ability to alleviate the symptoms of CNS disorders such
as schizophrenia and some mood disorders. Several previous studies have
shown that these agents can inhibit native T-type Ca channels in a
number of cell types (Enyeart et al., 1990, 1992; Arnoult et
al., 1998). In a systematic analysis of the cloned 1G, 1H, and
1I T-type Ca channel subtypes, we show here
that distinct classes of neuroleptics: the DPBPs (pimozide and
penfluridol), butyrophenones (haloperidol), and the diphenyldiperazine
derivative flunarizine, differentially affect neuronal T-type Ca channels.
Diphenybutylpiperidines are potent blockers of T-type
Ca channels
The DPBPs are a class of neuroleptic agents used in the treatment
of schizophrenia and related psychoses such as delusional disorder.
Pimozide is also used in the treatment of Tourette's syndrome, which
is a familial neurobehavioral disorder characterized by fluctuating
involuntary motor and/or vocal tics (Cohen et al., 1992). It is thought
that DPBPs owe much of their antipsychotic effectiveness to their
antagonism of dopamine receptors (Seeman et al., 1976). However,
several studies have shown they also interact with other nervous system
targets, including Ca channels (Enyeart et al., 1992; Sah and Bean,
1993). Our results have identified the DPBPs penfluridol and pimozide
as two of the most effective T-type Ca channel antagonists among the
available organic blockers. We find that the
Kd values for pimozide (39-60
nM) are very similar to published
Kd values for dopamine
D2 receptors (29 nM;
Richelson and Souder, 2000). Studies on neural crest-derived cell lines and adrenal zona fasciculata (AZF) cells have shown that penfluridol potently blocks T-type channels with IC50 values
of 224 nM (Enyeart et al., 1992) and 300 nM (Enyeart et al., 1993), respectively, whereas
pimozide inhibited T-type currents in the AZF cells and spermatogenic
cells with IC50 of 500 and 460 nM, respectively. Because the concentration of
permeant ion has been shown to affect piperidine blocking affinity
(Zamponi et al., 1996), the discrepancy between the blocking potency
observed in native T-type channels and that which we report in the
cloned channels may be explained by the different experimental
conditions used (10 mM Ca was used in native
channels vs 2 mM Ca/Ba used in our experiments).
To explore this possibility, experiments were performed in 10 mM external Ca for comparison. These experiments
showed that 1 µM penfluridol blocked
1G 71.6 ± 5.6% (n = 3)
in 10 mM Ca, whereas the same concentration
blocked T-type currents 90.9 ± 2.8 (n = 7)
1G in 2 mM Ca (data not
shown). Thus, the difference in Ca concentrations probably accounts for
only a small portion of the difference.
The potency of DPBPs as T-type Ca channel antagonists parallels their
potency as D2 receptor antagonists (cf. Gould et
al., 1983; this study). In contrast, butyrophenone and phenothiazine are much weaker Ca channel antagonists compared with
D2 dopamine receptor blockers (haloperidol
Kd values: D2
receptor, 2.6 nM, Richelson and Souder, 2000;
T-type channels, ~1200 nM; this study). Thus,
it is possible that at therapeutic doses DPBPs occupy receptor sites on
both T-type Ca channels and D2 dopamine
receptors, whereas butyrophenones like haloperidol bind almost
exclusively to D2 dopamine receptors. Clinically,
DPBPs offer an advantage over other drugs (e.g., butyrophenones)
because they are able to relieve the negative symptoms of schizophrenia
such as emotional withdrawal and poverty of speech that are resistant
to treatment with other classical neuroleptic drugs. The only known
pharmacological action that distinguishes DPBPs from other neuroleptics
such as the butyrophenones (e.g., haloperidol) is their high-potency
T-type Ca-antagonist activity. This raises the possibility that the
selective clinical improvement in negative symptoms observed for DPBPs
is attributable to their ability to block T-type calcium channels
(Gould et al., 1983; Snyder and Reynolds, 1985).
Neuroleptic agents preferentially bind to the
inactivated state
We find that the effects of all the neuroleptic drugs examined are
potentiated at more depolarized holding potentials. This suggests that
these drugs bind with higher affinity to the inactivated state of the
channels. Similar results in native cells showed that penfluridol (300 nM) and pimozide (500 nM) shifted the
steady-state inactivation of T-type currents by 8.2 and 10 mV,
respectively (Enyeart et al., 1993). In addition T-type currents from
spermatogenic cells have been shown to be blocked by pimozide in a
voltage-dependent manner (Arnoult et al., 1998). Flunarizine has also
been reported to have voltage-dependent inhibitory effects on many
native T-type currents; for example, 5 µM flunarizine
shifted the steady-state inactivation of T-type currents in rat
Purkinje neurons by 10 mV (Panchenko et al., 1993) and similarly in
mouse neuroblastoma cells (Wang et al., 1990) and guinea pig
ventricular myocytes (Tytgat et al., 1996). This voltage dependence of
block is particularly relevant in cells that fire action potentials
because more depolarized potentials would be predicted to enhance the
potency of blockade.
Flunarizine preferentially blocks 1G and
1I T-type channels
Flunarizine is a diphenyldiperazine derivative described as a Ca
channel antagonist (Tytgat et al., 1988; Akaike et al., 1989; Wang et
al., 1990), although it also displays moderate dopamine receptor
antagonist activity with reported Kd
values of 100-500 nM (Ambrosio and Stefanini,
1991; Brucke et al., 1995). Interestingly, these values are similar to
those found in our study for 1G and 1I T-type Ca channel blockade (~530 and 840 nM, respectively) (Table 1). Clinically,
flunarizine has been mainly prescribed to treat vertigo (Olesen, 1988),
migraine (Spierings, 1988), and epilepsy (Greenberg, 1987). The
mechanisms underlying the clinical efficacy of flunarizine are
considered to be related to modulation of the activity of neuronal Ca
channels (Moron et al., 1989). Flunarizine has been identified as a
moderately potent T-type Ca channel blocker in native cells such as
hypothalamic neurons (IC50 = 0.7 µM; Akaike et al., 1989). Similar effects of
flunarizine on T-type Ca currents have been observed in rat amygdaloid
neurons (Kaneda and Akaike, 1989), hippocampal CA1 pyramidal neurons
(Takahashi and Akaike, 1991), N1E-115 neuroblastoma cells (Wang et al.,
1990), and in Purkinje cells (Panchenko et al., 1993). In contrast,
T-type channels from guinea pig ventricular myocytes and rat calcitonin secreting C-cells display lower affinity for this compound
(Kd, ~10 µM
and IC50 ~10 µM,
respectively) (Enyeart et al., 1992; Tytgat et al., 1996). The
different blocking potencies of flunarizine on native T-type currents
could be explained by the recently described diversity in the molecular
composition of the T-type channels. Of particular relevance, we report
here that among the neuroleptic drugs tested, flunarizine displayed
preferential blockade of the 1G and
1I subtypes with
Kd values approximately five times
higher for the 1H channels (Table 1). A
similar difference in Kd was observed
when Ca was used as a charge carrier for 1H
(Kd, 3.04 µM).
The blockade of T-type Ca channels by flunarizine is consistent with
its depressive action on repetitive neuronal firing and may contribute
to its efficacy in suppressing seizure discharge (Bingmann and
Speckmann, 1989). The 1G T-type channel is
highly expressed in the thalamus (McRory et al., 2001) and may
represent the molecular target for flunarizine action.
In summary, we have characterized the effects of clinically important
neuroleptic agents on the 1G,
1H, and 1I T-type Ca channels expressed in the mammalian CNS. The results show that the
DPBPs antagonize T-type Ca channels with a potency similar to their
affinity for D2 dopamine receptors, and thus Ca
channel block may represent a significant contribution to the clinical efficacy of these agents. Flunarizine exhibited preferential block of
1G and 1I compared
with the 1H T-type Ca channel that may contribute to the differential therapeutic efficacy of this agent compared with DPBPs. Overall, the inhibitory effects of the DPBP neuroleptics on T-type Ca channels may underlie some of the clinical efficacy (and side effects) of antipsychotic treatments.
| |
FOOTNOTES |
Received May 30, 2001; revised Oct. 3, 2001; accepted Oct. 12, 2001.
This work was supported by a grant from Canadian Institutes for Health
Research (CIHR) (T.P.S.), fellowship support from the Human Frontiers
Science Program (C.M.S.), from the CIHR (J.E.M.), and from the Natural
Sciences and Engineering Research Council of Canada and Killam
Foundation (K.S.C.H.), and a CIHR Senior Scientist Award (T.P.S.).
Correspondence should be addressed to Dr. Terrance P. Snutch,
Biotechnology Laboratory, Room 237-6174, University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada V6T
1Z3. E-mail: snutch{at}zoology.ubc.ca.
| |
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ABSTRACT
INTRODUCTION
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