NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-N-
naphtyl cyclopropanecarboxylate dihydrochloride]: A New
Selective Inhibitor of T-Type Calcium Channels
Luping Huang, Brian M. Keyser, Tina M. Tagmose, J. Bondo Hansen, James T. Taylor,
Hean Zhuang, Min Zhang, David S. Ragsdale, and Ming Li
Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, Louisiana (L.H., B.M.K., J.T.T., M.L.);
Department of Medicinal Chemistry, Novo Nordisk A/S, Måløv, Denmark (T.M.T., J.B.H.); Department of Anesthesiology and
Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland (H.Z.); Department of Pharmacology, Medical College of
Virginia, Richmond, Virginia (M.Z.); and Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill
University, Montreal, Quebec, Canada (D.S.R.).
Received October 2, 2003; accepted December 5, 2003
Mibefradil is a Ca2?channel antagonist that inhibits both T-
type and high-voltage-activated Ca2?channels. We previously
showed that block of high-voltage-activated channels by mi-
befradil occurs through the production of an active metabolite
by intracellular hydrolysis. In the present study, we modified the
structure of mibefradil to develop a nonhydrolyzable analog,
clopropanecarboxylate dihydrochloride (NNC 55-0396), that
exerts a selective inhibitory effect on T-type channels. The
acute IC50of NNC 55-0396 to block recombinant ?1G T-type
channels in human embryonic kidney 293 cells was ?7 ?M,
whereas 100 ?M NNC 55-0396 had no detectable effect on
high-voltage-activated channels in INS-1 cells. NNC 55-0396
did not affect the voltage-dependent activation of T-type Ca2?
currents but changed the slope of the steady-state inactivation
curve. Block of T-type Ca2?current was partially relieved by
membrane hyperpolarization and enhanced at a high-stimulus
frequency. Washing NNC 55-0396 out of the recording cham-
ber did not reverse the T-type Ca2?current activity, suggesting
that the compound dissolves in or passes through the plasma
membrane to exert its effect; however, intracellular perfusion of
the compound did not block T-type Ca2?currents, arguing
against a cytoplasmic route of action. After incubating cells
from an insulin-secreting cell line (INS-1) with NNC 55-0396 for
20 min, mass spectrometry did not detect the mibefradil me-
tabolite that causes L-type Ca2?channel inhibition. We con-
clude that NNC 55-0396, by virtue of its modified structure,
does not produce the metabolite that causes inhibition of L-
type Ca2?channels, thus rendering it more selective to T-type
Voltage-gated Ca2?channels are transmembrane proteins
involved in the regulation of cellular excitability and intra-
cellular Ca2?signaling. Calcium channels have been divided
into various categories based on functional and pharmacolog-
ical criteria. High-voltage-activated (HVA) channels, which
have been further subdivided into L-, N-, P/Q-, and R-types,
require strong depolarizations for activation, whereas low-
voltage-activated or T-type channels activate over a much
more negative voltage range and exhibit unique inactivation
and deactivation kinetics (Armstrong and Matteson, 1985;
Catterall, 1998; Perez-Reyes, 1998). The main structural
component of the voltage-gated calcium channel is the ?1
This study was supported by American Heart Association Grant 0151047B
(to M.L.) and by Canadian Institutes of Health Research Grant MT13485 (to
Article, publication date, and citation information can be found at
ABBREVIATIONS: HVA, high-voltage-activated; HEK, human embryonic kidney; NNC 55-0395, (1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-
methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl valeroate dihydrochloride); NNC 55-0396, (1S,2S)-2-(2-(N-[(3-benzimidazol-
2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride; NNC 55-0397,
ride; PBS, phosphate-buffered saline; EI, electron impact; DMSO, dimethyl sulfoxide; G, conductance; MALDI-TOF, matrix-assisted laser
desorption ionization/time of flight instrument.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics
JPET 309:193–199, 2004
Vol. 309, No. 1
Printed in U.S.A.
at ASPET Journals on November 6, 2015
subunit, which forms the pore and the channel gates. Molec-
ular cloning has identified 10 ?1subtypes. ?1A–?1E and ?1S
encode HVA channels, whereas ?1G–?1I encode T-type chan-
nels. Pharmacological agents that act selectively on ?1sub-
types have been key in studying calcium channel function in
many physiological systems; however, a selective antagonist
of T-type Ca2?channels is not yet available.
Mibefradil, a tetralol derivative chemically distinct from
other Ca2?channel antagonists, has been reported to block
T-type Ca2?channel currents in many tissues, including
heart (Madle et al., 2001), brain (McDonough and Bean,
1998), and vascular smooth muscle (Bian and Hermsmeyer,
1993; Mishra and Hermsmeyer, 1994; Schmitt et al., 1995).
Mibefradil was also reported to block HVA Ca2?channels,
including ?1A, ?1B, ?1C, and ?1E (Bezprozvanny and Tsien,
1995; Jime ´nez et al., 2000). We previously demonstrated that
mibefradil potently blocked HVA Ca2?channels in a rat
insulin-secreting cell line (INS-1 cells) via a mechanism in-
volving intracellular hydrolysis of mibefradil to produce an
active metabolite (Wu et al., 2000).
The cardiovascular effects of mibefradil are interesting; for
instance, it decreases heart rate without a negative inotropic
effect (Osterrieder and Holck, 1989; Cremers et al., 1997),
and its action is not associated with the reflex activation of
neurohormonal and sympathetic systems (Ernst and Kelly,
1998). These properties differ from other clinically important
Ca2?antagonists such as nifedipine, diltiazem, and vera-
pamil, which in the heart selectively inhibit L-type (?1C)
Ca2?channels. However, it is unclear whether the unique
effects of mibefradil are caused by the blockade of T-type
Ca2?channels since mibefradil also blocks the L-type Ca2?
currents in the cardiomyocytes (Leuranguer et al., 2001).
Therefore, identifying a more selective T-type Ca2?channel
antagonist will be useful for the study of cardiac voltage-
gated calcium channels and may promote the development of
a new class of therapeutically beneficial compounds.
In a previous study, we demonstrated that hydrolysis of
the ester side chain of mibefradil resulted in a compound
(des-methoxyacetyl mibefradil or Ro 40-5966) that exhibited
an L-type Ca2?channel-specific inhibitory effect (Wu et al.,
2000). We thus proposed that modifications in this ester side
chain, which decreased hydrolysis, might result in com-
pounds with a lower potency in blocking L-type Ca2?chan-
nels and selective action on T-type channels. To test this
hypothesis, we examined the effects of several novel mibe-
fradil derivates on T-type and HVA Ca2?currents in whole-
cell voltage-clamp recordings. We used HEK 293 cells stably
transfected with the ?1G subtype of T-type Ca2?channel
(HEK/?1G) and INS-1 cells to test the effects of our new
compounds on T-type and HVA Ca2?channels, respectively.
INS-1 cells express a variety of HVA Ca2?currents, includ-
ing P/Q-, N-, L-, and R-types (Horvath et al., 1998), with ?1D
contributing most of the currents (Liu et al., 2002; Huang et
Materials and Methods
Cell Culture. INS-1 cells, an insulin-secreting cell line derived
from rat pancreatic ?-cells (Asfari et al., 1992) were cultured in
RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml
penicillin, 100 ?g/ml streptomycin, and 50 ?M mercaptoethanol in
an atmosphere of 5% CO2in air at 37°C for 2 to 5 days before
Creation of HEK 293 Cell Lines Stably Expressing Recom-
binant T-Type Ca2?Channels. An ?1G cDNA originally cloned
from rat pancreatic ?-cells (Zhuang et al., 2000) in vector pcDNA3.1
hygro(?) (Invitrogen, Carlsbad, CA) was transfected into HEK cells
using the FuGENE kit (Roche Diagnostics, Indianapolis, IN). Cell
lines stably expressing Cav3.1 were obtained after transfection using
standard cell cloning techniques (Freshney, 1983). HEK 293 cells
stably transfected with ?1G cDNA (HEK 293/?1G) were incubated in
Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 100 ?g/ml streptomycin, and 0.5
mg/ml hygromycin-B in an atmosphere of 5% CO2in air at 37°C for
2 to 5 days before recording.
Electrophysiological Recording. Whole-cell recordings were
carried out by the standard “giga-seal” patch-clamp technique. The
whole-cell recording pipettes were made of hemocapillaries (Warner
Instrument, Hamden, CT), pulled by a two-stage puller (PC-10;
Narishige, Greenvale, NY), and heat-polished with a microforge
(MF-200; World Precision Instruments, Sarasota, FL) before use.
Pipette resistance was in the range of 2 to 5 M? in our internal
solution. The recordings were performed at room temperature (22–
25°C). Electrical currents were recorded using an EPC-9 patch-
clamp amplifier (HEKA, Lambrecht/Pfalz, Germany) and filtered at
2.9 kHz. Data were acquired with PULSE/PULSEFIT software
(HEKA). Voltage-dependent currents were corrected for linear leak
and residual capacitance by using an on-line P/n subtraction para-
digm. In the whole-cell configuration, T-type Ca2?currents were
recorded at ?20 or ?10 mV when the holding potential was ?70 mV.
The HVA Ca2?current was measured at 10 mV, with a holding
potential of ?40 mV.
Solutions. The extracellular solution used in the whole-cell Ca2?
current recording contained 10 mM CaCl2, 110 mM tetraethylam-
monium-Cl, 10 mM CsCl, 10 mM HEPES, 40 mM sucrose, 0.5 mM
3,4-diaminopyridine, pH 7.3. The intracellular solution contained
130 mM N-methyl-D-glucamine, 20 mM EGTA (free acid), 5 mM
HEPES, 6 mM MgCl2, and 4 mM Ca(OH)2, with pH adjusted to 7.4
with methanesulfonate. Mg-ATP (2 mM) was included in the pipette
solution to minimize rundown of L-type Ca2?currents. For perforat-
ed-patch experiments, 200 ?g/ml nystatin was used. The pipette was
filled with nystatin-containing intracellular solution, and gentle suc-
tion was used to achieve gigaohm resistance. The access resistance
gradually decreased within 5 min after the gigaohm-seal formation,
and then currents were recorded after stabilization. The extracellu-
lar solution contained 26 mM sucrose, 130 mM tetraethylammoni-
um-Cl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, pH
7.3. The pipette solution contained 65 mM CsOH, 65 mM Cs-meth-
anesulfonate, 20 mM sucrose, 10 mM HEPES, 10 mM MgCl2, and 1
mM Ca(OH)2, with pH 7.4.
Mass Spectrometric Analysis. Mass spectrometric analysis was
performed on a PerSeptive Voyager-DE MALDI-TOF instrument
(Applied Biosystems, Foster City, CA). Cultured INS-1 cells were
treated with 20 ?M NNC 55-0396 for various lengths of time under
each experimental condition. After incubation, cells were washed
with PBS three times, scraped into Eppendorf tubes with 1 ml of
PBS, and centrifuged at 1000g for 5 min. The cell pellets were
collected and redissolved by sonication (10 s) in 0.5 ml of PBS for
mass spectrometric analysis. The sample was taken up in ?-cyano-
4-hydroxcinnamic acid (Aldrich, Milwaukee, MI), which was used as
the matrix. Ten microliters of each sample were mixed with 70 ?l of
the matrix. One microliter of the mixture was spotted on a plate for
analysis on the MALDI-TOF instrument. Several positive ion spec-
tra were recorded in the mass range m/z 820 to 99 at a mass
resolution of 1000 and a scan speed of 2 s/decade. For NNC 55-0396,
m/z 492 was the dominant ion (M ? H). For calibration, a standard
solution of 50 ?M NNC 55-0396 was subjected to mass spectrometric
analysis. The relative amount of NNC 55-0396 was determined by
calibrating the intensities of NNC 55-0396 with the intensity of
Huang et al.
at ASPET Journals on November 6, 2015
Statistics and Curve Fitting. Nonlinear regression analysis
was used to fit concentration-response data to a sigmoid relation-
ship, Y ? 100/(1 ? 10ˆ((log IC50? X) slope), where slope is the Hill
slope parameter, IC50is the concentration producing 50% blockage,
and X is the drug concentration. Voltage-dependent activation and
steady-state inactivation curves were generated by normalizing the
currents with the maximal amplitude in each cell and fitting the data
with the Boltzmann equation, 1/(1 ? exp((V ? V1/2)/k), where k
represents the slope and V1/2represents the voltage corresponding to
half-activation of the channels. Student’s t test was used to compare
V1/2and k values determined from fits of the data with this equation.
The data fitting and statistical analysis were performed with Prism
version 4 (GraphPad Software, San Diego, CA). In the figures where
the data are presented as symbols and error bars, the values are
mean ? S.E.M.
General Chemical Procedure. Reagents, starting materials,
and solvents were purchased from common commercial suppliers
and were used as received. All dry solvents were dried over molecular
sieves (0.3 or 0.4 nm). Evaporation was carried out on a rotary
evaporator at bath temperatures ?40°C and under appropriate vac-
uum. Flash chromatography was carried out on a Biotage FLASH 40
(Biotage AB, Uppsala, Sweden) using Biotage FLASH columns (KP-
SIL 60 Å particle size, 32–63 ?M). Melting points were determined
with a Bu ¨chi B545 apparatus (Bu ¨chi, Flawil, Switzerland) and are
uncorrected. Proton NMR spectra were recorded at ambient temper-
ature using a Bruker AVANCE DPX 200 (Bruker, Newark, DE) (200
MHz) and DPX 300 (300 MHz), with tetramethylsilane as an inter-
nal standard for proton spectra. Chemical shifts are given in ppm (?),
and splitting patterns are designated as follows: s, singlet; d, dou-
blet; dd, double doublet; t, triplet; dt, double triplet; q, quartet; quint,
quintet; m, multiplet; and br, broad. The 70-electron-volt EI solid
mass spectra were recorded on a Finnigan MAT TSQ 70 mass spec-
trometer (Thermo Finnigan, San Jose, CA). Reactions were followed
by thin layer chromatography performed on silica gel 60 F254
(Merck, Darmstadt, Germany) or ALUGRAM SIL G/UV254(Mach-
erey-Nagel, Du ¨ren, Germany) thin layer chromatography aluminum
Synthesis of NNC 55-0396. Methoxyacetic acid (2(S)-[2-[N-[3-(2-
pyl-1,2,3,4-tetrahydro-2-naphthyl ester dihydrochloride) (mibefradil,
0.570 g) in ethanol (96%, 5 ml) and aqueous sodium hydroxide (1 N,
5 ml) was refluxed for 2 h. The cold reaction mixture was concen-
trated in vacuo. The residue was partitioned between water and
dichloromethane. The aqueous layer was extracted with dichlo-
romethane. The combined organic layers were dried with sodium
sulfate and concentrated to give 2-(2-[[3-(1-benzimidazol-2-yl)-
2-naphthalinol as a clear syrup (100%, 0.43 g).1H NMR (CDCl3): ?
7.57 (br, 2H); 7.23 (m, 2H); 6.97 (m, 1H); 6.58 (m, 2H); 3.07–2.83 (m,
3H); 2.75 (m, 1H); 2.6 (m, 4H); 2.5–2.2 (s ? m, 3H ? 3H); 2.06 (quint,
2H); 1.81 (br dd, 1H); 1.50 (m, 2H); 1.20 (d, 3H); 0.53 ppm (d, 3H).
fluoro-1-isopropyl-1,2,3,4-tetrahydro-2-naphthalinol (0.110 g) was
dissolved in 1 ml of dichloromethane. Diisopropylethylamine (0.045
ml) and 0.071 ml of cyclopropanecarbonyl chloride was added. After
stirring for 19 h, the reaction mixture aqueous saturated sodium
hydrogencarbonate was added. The aqueous layer was extracted
twice with dichloromethane. The combined organic layers were dried
with sodium sulfate and concentrated in vacuo. The residue was
purified by flash chromatography using dichloromethane/methanol
6:1 as an eluent to give the free base as a syrup. This product was
dissolved in ethanol, and aqueous hydrochloride (1 N, 0.38 ml) was
added. After stirring for 30 min, the mixture was concentrated. The
residue was crystallized from ethyl acetate to give the title compound
as a white powder (34%, 50 mg). Mp: 134 to 141°C. EI SP/MS: 491
(M?).1H NMR (DMSO—d6): ? 7.77 (m, 2H); 7.52 (m, 2H); 7.07 (m,
1H); 6.96 (broad d, 2H); 3.3 (m, 2H); 3.2 (m, 4H); 3.0 (m, 2H); 2.9 (m,
1H); 2.67 (s, 3H); 2.45 (m, 1H); 2.38 (m, 2H); 2.15–1.45 (m, 4H); 1.57
(m, 1H); 1.04 (d, 3H); 0.90 (m, 4H); 0.48 ppm (d, 3H).
Synthesis of (1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-
pyl-2-naphtyl valeroate dihydrochloride (NNC 55-0395). NNC
55-0395 was prepared by an analogous procedure: Mp 118 to 121°C.
EI SP/MS: 507 (M?).1H NMR (DMSO-d6): ? 7.75 (m, 2H); 7.49 (m,
2H); 7.08 (m, 1H); 6.96 (br d, 2H); 3.15 (m, 4H); 2.95 (m, 3H); 2.7 (s,
3H); 2.48 (dt, 2H); 2.23 (m, 2H); 2.0 (m, 4H); 1.50 (p, 2H); 1.35 (quint,
2H); 1.02 (d, 3H); 0.90 (t, 3H); 0.35 ppm (d, 3H).
Synthesis of (1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-
pyl-2-naphtyl isobutyrate dihydrochloride (NNC 55-0397).
NNC 55-0397 was prepared by an analogous procedure: Mp 114 to
117°C. EI SP/MS: 493 (M?).1H NMR (DMSO-d6): ? 7.77 (m, 2H);
7.52 (m, 2H); 7.07 (m, 1H); 6.96 (br d, 2H); 3.43 (m, 1H); 3.3- 3.05 (m,
5H); 2.97 (m, 2H); 2.88 (m, 1H); 2.70 (s, 3H); 2.61 (m, 1H); 2.45 (m,
1H); 2.3 (m, 2H);2.15–1.85 (m, 4H); 1.15 (d, 6H); 1.00 (d, 3H); 0.45
ppm (d, 3H).
Synthesis of NNC 55-0396 and Other Mibefradil An-
alogs. We synthesized three analogs of mibefradil by replac-
ing the ester chain (methoxyacetyl) group NNC 55-0395,
NNC 55-0396, and NNC 55-0397 (Fig. 1). All compounds
were synthesized from mibefradil in two steps. Alkaline hy-
drolysis of mibefradil and subsequent treatment with
valeroyl chloride, cyclopropanecarbonyl chloride, and isobu-
tyryl chloride gave the desired compounds NNC 55-0395,
NNC 55-0396, and NNC 55-0397 upon workup, respectively.
Pharmacological-Screening Effect of NNC 55-0395,
NNC 55-0396, and NNC 55-0397 on T-Type and HVA
Ca2?Currents. We used perforated whole-cell patch-clamp
to examine the effects of NNC 55-0395, NNC 55-0396, and
NNC 55-0397 on HVA Ca2?current in INS-1 cells. In these
experiments, the resting membrane potential was held at
?40 mV to eliminate T-type Ca2?current. The inhibitory
potency of the mibefradil analogs on HVA Ca2?current was
examined at dosages of 0.1, 1, 10, and 100 ?M approximately
10 min after the drug perfusion. The results showed that
both NNC 55-0395 and NNC 55-0397 had an inhibitory effect
on HVA Ca2?currents at 100 ?M (Fig. 2, A and C), whereas
no inhibition on the HVA Ca2?current was detected with
NNC 55-0396 at the same concentration (Fig. 2B).
Fig. 1. Synthesis of NNC 55-0396 and other analogs from mibefradil and
chemical structures of NNC 55-0395, NNC 55-0396, and NNC 55-0397.
The side chain of mibefradil inside the dashed box is replaced by the new
structures indicated by arrows.
Selective T-Type Ca2?Channel Blocker
at ASPET Journals on November 6, 2015
Next, we determined the effects of NNC 55-0395, NNC
55-0396, and NNC 55-0397 on T-type Ca2?current. These
experiments were conducted by measuring the effects of the
compounds on whole-cell current in HEK 293/?1G cells. The
dose-dependent inhibition of compounds NNC 55-0395, NNC
55-0396, and NNC 55-0397 on T-type Ca2?current was ex-
amined at dosages ranging from 1 to 100 ?M. NNC 55-0396
and NNC 55-0397 blocked more than 50% of the T-type Ca2?
current at 8 ?M (Fig. 2, E and F), whereas NNC 55-0395
inhibited less than 50% of the T-type Ca2?current at 64 ?M
Both NNC 55-0395 and NNC 55-0397 blocked HVA Ca2?
channel currents in our screening experiments and were thus
eliminated from further characterization. Compound NNC
55-0396, which blocked T-type Ca2?current but not HVA
Ca2?currents, was selected for subsequent analysis.
Characterization of the Inhibitory Effects of NNC
55-0396 on T-Type Ca2?Current. To further characterize
NNC 55-0396, we used whole-cell patch-clamp and a bath
perfusion system to examine its dose-dependent effects on
T-type and HVA Ca2?currents. At 8 ?M, NNC 55-0396
reduced more than 50% of the peak of T-type Ca2?current
compared with the control in HEK 293/?1G cells (Fig. 3A);
however, NNC 55-0396 at 100 ?M did not block the HVA
Ca2?current in INS-1 cells (Fig. 3B), whereas this HVA Ca2?
current could be inhibited by 10 ?M nifedipine, a selective
L-type Ca2?channel blocker (Fig. 3C). Pooled data of the
effects of NNC 55-0396 on T-type and HVA Ca2?currents are
shown in Fig. 3D. After bathing HEK 293/?1G cells with
NNC 55-0396 at 8 ?M or bathing INS-1 cells with NNC
55-0396 at 100 ?M for over 8 min, T-type Ca2?currents were
inhibited by 60%, whereas no decrease in the HVA Ca2?
current was observed.
The inhibitory potency of NNC 55-0396 on T-type Ca2?
currents in HEK 293/?1G was also compared with that of
mibefradil, as shown in Fig. 3E. By fitting the data with a
sigmoid dose-response relationship equation, NNC 55-0396
and mibefradil blocked T-type Ca2?current with IC50values
of 6.8 and 10.08, respectively. This result suggests that NNC
55-0396 retains potency similar to mibefradil as an inhibitor
of T-type Ca2?current.
Long-term exposure of HEK/?1G cells to NNC 55-0396
showed a decrease in current density of T-type Ca2?chan-
nels. Using whole-cell patch-clamp, we measured T-type
Ca2?current density in the HEK/?1G cells that had bathed
in the extracellular solutions containing 0, 10, 100, and 1000
nM NNC 55-0396 for 30 to 60 min (Fig. 3F). All peak-current
amplitudes and slow capacitances were obtained during the
first minute after breaking in; thus, the effect of the frequen-
cy-dependent inhibitory effect was minimal. Our data
showed a slow yet more potent inhibitory effect of NNC
55-0396 on the T-type Ca2?currents.
Figure 4A illustrates that there was no significant differ-
ence in the conductance-voltage relationship (G/V) curves of
Fig. 2. Pharmacological-screening effect of NNC 55-0395, NNC 55-0396, and NNC 55-0397 on HVA and T-type Ca2?currents. A, B, and C,
dose-dependent effects of the three compounds on HVA currents recorded with perforated whole-cell patch-clamp in INS-1 cells with a holding
potential of ?40 mV and a test pulse at 10 mV for 200 ms for every 30 to 40 s. The relative current values were obtained by normalizing the current
amplitudes with respect to those recorded under the drug-free condition. D, E, and F, dose-dependent effects of the three compounds on T-type Ca2?
currents recorded with whole-cell patch-clamp in HEK 293/?1G cells with a holding potential of ?70 mV and a testing potential of ?20 mV for 200
ms (n ? 3–5 for each set of data).
Huang et al.
at ASPET Journals on November 6, 2015
T-type Ca2?currents between the control cells and cells
incubated with 8 ?M NNC 55-0396. The conductance (G) was
calculated from the current (I) divided by the driving force
(Vdrive? Vmembrane? Vreversal), where the Vreversalwas hy-
pothetically assigned as 70 mV. Curves were generated by
fitting the data with the Boltzmann equation. The V1/2and k
values are ?32.61 ? 1.0 and 4.16 ? 0.9 for the controls (n ?
3) and ?32.12 ? 0.8 and 4.33 ? 0.73 for cells incubated with
NNC 55-0396 (n ? 3), respectively. The steady-state inacti-
vation curves of T-type Ca2?current were also generated
with the Boltzmann equation (Fig. 4B). The V1/2values are
?59.21 ? 0.38 and ?62.84 ? 0.94 for the controls (n ? 4) and
cells incubated with NNC 55-0396 (n ? 6), respectively. The
slope k values are 4.92 ? 0.33 with a 95% confidence interval
between 4.16 to 5.69 for the controls and 7.95 ? 0.72 with a
95% confidence interval between 6.31 to 9.58 for cells incu-
bated with NNC 55-0396, respectively. Therefore, the steady-
state inactivation curve of T-type Ca2?current is flattened
when NNC 55-0396 is present.
State-Dependent Block of T-Type Ca2?Current by
NNC 55-0396. The inhibitory mechanism of NNC 55-0396 on
T-type Ca2?current was further investigated by testing the
effect of changing the holding potential and stimulation fre-
quency. As shown in Fig. 5A, in the absence of NNC 55-0396,
changing the holding potential from ?120 to ?80 mV did not
alter the peak amplitude of T-type Ca2?current in an HEK
293/?1G cell, elicited by depolarizations to ?10 mV every
10 s. In contrast, after the addition of 8 ?M NNC 55-0396,
switching the membrane potential from ?80 to ?120 mV
caused an increase in the current amplitude, suggesting a
partial relief of block at the more hyperpolarized holding
potential. Similarly, increasing the pulse rate from 0.05 to
0.5 Hz had little effect on current amplitude without NNC
Fig. 4. Effects of NNC 55-0396 on voltage-dependent activation (G/V) and
steady-state inactivation of T-type Ca2?channel. A, voltage-dependent
activation (G/V) curves of T-type Ca2?channel with (solid circles) and
without (open circles) the presence of 8 ?M NNC 55-0396 were fit by the
Boltzmann equation (n ? 3). B, steady-state inactivation curves of T-type
Ca2?channel with (solid circles) and without (open circles) the presence
of 8 ?M NNC 55-0396 were fit by the Boltzmann equation (n ? 6).
Steady-state inactivation was determined by applying a prestimulating
pulse of 1.5 s at various voltages immediately before the test pulse at ?20
mV. For both A and B, currents were recorded in HEK 293/?1G cells, and
the cell membrane was held at ?70 mV.
Fig. 3. The effects of NNC 55-0396 on T-type and HVA Ca2?channels. A, current traces of T-type Ca2?currents in an HEK 293/?1G cell before and
after application of 8 ?M NNC 55-0396. The holding potential was ?70 mV, and the test potential was ?20 mV. B, current traces of HVA Ca2?currents
in an INS-1 cell before and after 100 ?M NNC 55-0396. The currents were recorded at 10 mV for 200 ms when held at ?40 with perforated whole-cell
patch-clamp. The traces were filtered at 1 kHz. C, Ca2?current was inhibited by 10 ?M nifedipine in an INS-1 cell. The currents were measured at
10 mV before and after nifedipine (Nif) administration with a holding potential of ?70 mV. D, time-dependent effects of NNC 55-0396 on HVA and
T-type Ca2?currents. The open circles represent relative currents of HVA Ca2?channels recorded in INS-1 cells before and after 100 ?M NNC 55-0396
administration (n ? 6), whereas the solid circles represent relative currents recorded in HEK 293/?1G cells before and after 8 ?M NNC 55-0396
administration (n ? 4). NNC 55-0396 was applied to the bath 3 min after the beginning of the recording. E, dose-dependent inhibitory effects of NNC
55-0396 (open circles, n ? 6) and mibefradil (solid circles, n ? 4) on T-type Ca2?currents recorded in HEK/?1G cells. The smooth lines are consistent
with the Hill equation, with IC50values of 6.8 and 10.08 ?M for NNC 55-0396 and mibefradil, respectively. The Hill slopes are ?2.39 and ?4.26 for
NNC 55-0396 and mibefradil, respectively. F, comparison of current densities (pA/pF) among HEK/?1G cells incubated in various concentrations of
NNC 55-0396 for 30 to 60 min. The peak-current amplitudes were measured at ?20 mV when held at ?70 mV (n ? 7–11 for each set of data). The
asterisk represents p ? 0.01 of the data compared with the control (0 nM NNC 55-0396).
Selective T-Type Ca2?Channel Blocker
at ASPET Journals on November 6, 2015
55-0396, whereas the rate of inhibition of T-type Ca2?cur-
rent was accelerated by increasing the frequency of stimula-
tion in the presence of the drug (Fig. 5B). Similar results of
both voltage-dependent and frequency-dependent blockade
were observed in four experiments.
Treated INS-1 Cells. Previously, we found that mibefradil
blocked both T-type and HVA Ca2?current in INS-1 cells
(Wu et al., 2000). After entering the cells, mibefradil is bro-
ken down into metabolites. One of them is des-methoxyacetyl
mibefradil, which exerts an inhibitory effect on HVA Ca2?
channels by acting on the channels from inside the cell. To
investigate why NNC 55-0396 and mibefradil have different
effects on HVA Ca2?channels, we used mass spectrometric
analysis to examine whether des-methoxyacetyl mibefradil
was produced intracellularly when the cells were treated
with NNC 55-0396. Figure 6A shows a section of mass spec-
trum of molecules in INS-1 cells that had been incubated
with NNC 55-0396 for 20 min. Notably, no des-methoxyacetyl
mibefradil (which peaks at 424 m/z) was detected in this
preparation. This finding suggests that, unlike mibefradil,
the compound NNC 55-0396 is not hydrolyzed into des-me-
thoxyacetyl mibefradil. Thus, NNC 55-0396 does not inhibit
HVA Ca2?channels as mibefradil does. The amount of NNC
55-0396 (which peaks at 492 m/z), however, increased in the
cells for longer periods than 10 min as shown in Fig. 6B.
Accessibility of NNC 55-0396 on the T-Type Ca2?
Channel. The slow onset of NNC 55-0396 inhibition on T-
type Ca2?current and the increasing NNC 55-0396 accumu-
lation in the cells with time suggest that this compound
dissolves in or passes through the plasma membrane to exert
its effect. To test this hypothesis, we examined the revers-
ibility of the NNC 55-0396 blockade of T-type Ca2?current.
Whole-cell currents were evoked in HEK 293/?1G cells by
test pulses to ?10 mV from a holding potential of ?70 mV.
After establishing a steady current, small volumes of 8 ?M
NNC 55-0396 were delivered in close proximity to the record-
ing cell with a quartz capillary positioned by a micromanip-
ulator. The drug was washed out after more than a 50%
inhibition of T-type Ca2?current was achieved. As shown in
Fig. 7A, the blockade of the T-type Ca2?current by NNC
55-0396 was poorly reversible during a 10-min washout pe-
riod in these experiments. Since the NNC 55-0396 blocks
T-type Ca2?current at a relatively slow rate and is poorly
reversible by washing out, the drug binding site of the chan-
nel may be within transmembrane or intracellular domains
of the channel.
If the site of action of NNC 55-0396 is on the intracellular
side of the membrane, we would expect that intracellular
perfusion of the drug would effectively block T-type Ca2?
current. To test this possibility, whole-cell currents were
recorded in HEK 293/?1G cells with 8 ?M NNC 55-0396
added to the pipette solution. As shown in Fig. 7B, there was
Fig. 7. Washout and intracellular application of NNC 55-0396. A, inhib-
itory effect of 8 ?M NNC 55-0396 on HEK/?1G T-type Ca2?currents was
not reversed after perfusion with the drug-free extracellular solution (n ?
3). B, effect of intracellular application of NNC 55-0396 on HEK/?1G
T-type Ca2?currents. The currents were recorded by the whole-cell
patch-clamp with a pipette solution containing 8 ?M NNC 55-0396 (solid
circles, n ? 10) or a drug-free solution (open circles, n ? 6). All recordings
were conducted at a holding potential of ?70 mV and a test pulse of ?20
Fig. 5. State-dependent inhibition of T-type Ca2?channels by NNC
55-0396. A, whole-cell Ca2?currents were measured at ?10 mV when
held at ?120 and ?80 mV, as indicated by the bar above the graph. The
pulses were applied every 10 s. B, whole-cell Ca2?currents were mea-
sured at ?10 mV when held at ?70 mV at stimulation frequencies of 0.05
and 0.5 Hz, as indicated by the bar above the graph. NNC and the arrows
indicate the time when NNC 55-0396 was perfused into the bath.
Fig. 6. Mass spectrometric analysis of INS-1 cells treated with NNC
55-0396. A, no ester side chain hydrolytic metabolite (peak is predicted at
424 m/z) was detected in INS-1 cells after 30 min of incubation with 20
?M NNC 55-0392, which peaked at 492 m/z. B, time-related relative
intensity of NNC 55-0396 in INS-1 cells. Mass spectrometry was per-
formed on the cells that had been incubated in 20 ?M NNC 55-0396 for
various time periods and then washed three times. The relative intensity
is presented as the ratio of the energy power of the laser between the
NNC 55-0396 found inside the cells and the standard solution containing
50 ?M NNC 55-0396 (n ? 4).
Huang et al.
at ASPET Journals on November 6, 2015
no significant difference between the current in HEK 293/
?1G cells with (n ? 10) and without (n ? 6) NNC 55-0396
included in the pipette solution. For example, at the 10-min
timepoint, the p value is 0.12 with two-tailed unpaired Stu-
dent’s t test analysis. These data are inconsistent with the
idea that the site of action of NNC 55-0396 is on the cyto-
plasmic face of the T-type Ca2?channel. Thus, NNC 55-0396
appears to exhibit a relatively slow and wash-resistant bind-
ing to a site, perhaps within the transmembrane domains of
Mibefradil blocks both T-type Ca2?and HVA Ca2?chan-
nels. Although mibefradil appears to act directly on T-type
channels, we previously reported that its effect on HVA chan-
nels is not direct but instead involves cell permeation and
hydrolysis to an active metabolite that acts from the cyto-
plasmic side of the membrane (Wu et al., 2000). In the
present study, we developed a new compound, NNC 55-0396,
that has an inhibitory effect on T-type Ca2?current in HEK/
?1G cells but is not hydrolyzed to an active metabolite and
does not block HVA Ca2?currents in INS-1 cells. Thus, NNC
55-0396 is a selective inhibitor for T-type Ca2?channels. Our
data also suggest that the effect of NNC 55-0396 on T-type
channels is state-dependent; i.e., block is partially relieved by
membrane hyperpolarization and enhanced by high-fre-
quency channel activation.
Our previous findings (Wu et al., 2000) suggest that the
methoxyacetyl group of mibefradil is a critical determinant
for binding to HVA channels since its hydrolysis is the crit-
ical step in the binding of the compound to HVA channels. In
contrast, this moiety may play only a modest role in inter-
acting with the receptor domain of T-type Ca2?channels
since all three alternations—NNC 55-0395, NNC 55-0396,
and NNC 55-0397—retain the capability of blocking T-type
Ca2?current, although with varying potency. Our study
indicates that modification in the methoxyacetyl group of
mibefradil can improve its selectivity for T-type Ca2?chan-
nels over the HVA Ca2?channels without sacrificing the
potency of T-type Ca2?channel antagonism.
The primary objective of this study is to examine the struc-
ture-selectivity relationship of a small group of mibefradil
derivatives. Therefore, we chose HEK/?1G as a clean model
system. We do not know how effectively NNC 55-0396 will
block other T-type Ca2?channels, nor do we know its potency
on ?1G when other auxiliary subunits are present. However,
we speculate that NNC 55-0396 will block ?1H and ?1I, for it
has been shown that the IC50values of mibefradil blocking
?1G, ?1H, and ?1I are similar (Martin et al., 2000). In sum-
mary, our findings represent an important step toward de-
velopment of a specific T-type Ca2?inhibitor and thus have
potentially important scientific and therapeutic implications.
We thank Dr. B. Z. Peterson for constructive criticism on this
work. The mass spectrometric analyses were carried out in the
Louisiana State University Health Sciences Center New Orleans
Core Laboratories (New Orleans, LA).
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Address correspondence to: Dr. Ming Li, Department of Pharmacology
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Selective T-Type Ca2?Channel Blocker
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