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Vol. 62, Issue 4, 836-846, October 2002
-Subunit
Departments of Molecular and Cellular Pharmacology (Y.J., K.S., A.Y., A.H., S.O., K.M.), and Organic and Medicinal Chemistry (M.U., T.O.), Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan; and Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan (M.U., T.O.)
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Abstract |
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Effects of pimaric acid (PiMA) and eight closely related compounds on
large-conductance K+ (BK) channels were examined using
human embryonic kidney (HEK) 293 cells, in which either the
subunit
of BK channel (HEKBK
) or both
and
1 (HEKBK
1) subunits
were heterologously expressed. Effects of these compounds (10 µM) on
the membrane potential of HEKBK
1 were monitored by use of
DiBAC4(3), a voltage-sensitive dye. PiMA, isopimaric acid,
sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid,
and dihydroisopimarinol induced substantial membrane hyperpolarization.
The direct measurement of BK
1 opening under whole-cell voltage
clamp showed that these six compounds activated BK
1 in a very
similar concentration range (1-10 µM); in contrast, abietic acid,
sclareol, and methyl pimarate had no effect. PiMA did not affect the
charybdotoxin-induced block of macroscopic BK
1 current. Single
channel recordings of BK
1 in inside-out patches showed that 10 µM PiMA did not change channel conductance but significantly
increased its open probability as a result of increase in sensitivity
to Ca2+ and voltage. Because coexpression of the
1
subunit did not affect PiMA-induced potentiation, the site of action
for PiMA is suggested to be BK
subunit. PiMA was selective to BK
over cloned small and intermediate Ca2+ activated
K+ channels. In conclusion, PiMA (>1 µM) increases
Ca2+ and voltage-sensitivity of BK
when applied from
either side of the cell membrane. The marked difference in potency as
BK channel openers between PiMA and abietic acid, despite only very
small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BK
activation.
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Introduction |
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Large
conductance Ca2+ activated
K+ (BK) channels are expressed in many different
types of excitable cells and have significant physiological roles in
the regulation of frequency of firing, action potential repolarization,
and/or afterhyperpolarization (for reviews, see Vergara et al., 1998
;
Kaczorowski and Garcia, 1999
). In addition, BK channels are often a key
factor for the negative feedback control of intracellular
Ca2+ concentration
([Ca2+]i) and save cells
from Ca2+ overload during pathophysiological
conditions (Lawson, 2000
). Excess intracellular
Ca2+ is a major cause of neuronal cell death in
the setting of brain ischemia after stroke. The activation of BK
channels by elevation of
[Ca2+]i after
Ca2+ influx from outside and/or
Ca2+ release from endoplasmic reticulum
hyperpolarizes the cell and down-regulates the activity of
voltage-dependent Na+ and
Ca2+ channels in neuronal cells. In smooth muscle
cells, activation of BK channels by spontaneous
Ca2+ release (Ca2+ sparks)
from sarcoplasmic reticulum (Nelson et al., 1995
; Jaggar et al., 2000
)
is thought to be an essential regulator of resting membrane potential.
In vascular smooth muscle of spontaneous hypertensive rats, the
expression of BK channel is enhanced (Liu et al., 1997
) and the
deficiency of BK channel activity in
1 subunit gene-lacking mice
results in systemic hypertension (Brenner et al., 2000b
). BK channel
activation by the Ca2+-induced
Ca2+ release during excitation-contraction
coupling also significantly contributes to action potential
repolarization/afterhyperpolarization in smooth muscle cells (Imaizumi
et al., 1998
; Ohi et al., 2001
).
For these reasons, agents that enhance BK channel activity (BK channel
openers) may be effective in protecting neurons from damage after an
ischemic stroke and/or suppressing excess activity of smooth muscle
tissues (Lawson, 2000
; Shieh et al., 2000
). Many compounds have been
reported to be BK channel openers; dehydrosoyasaponin-I, maxikdiol,
L-735,334, NS-4, NS-1608, NS-1619, niflumic acid, cromakalim, nitrendipine, BMS-204352, NS-8, CGS-7181, CGS-7184, etc. (for review,
see Starrett et al., 1996
; Coghlan et al., 2001
). 17
-Estradiol (Valverde et al., 1999
) and epoxyeicosatrienoic acids (Fukao et al.,
2001
) may be endogenous BK channel openers. Moreover, some transmitters
and hormones can also enhance BK channel activity via activation of
cAMP and cGMP dependent protein kinases (for review, see Vergara et
al., 1998
).
BK channels consist of channel forming
subunits and accessory
subunits arranged in tetramers (Vergara et al., 1998
). Each
subunit
interacts with N-terminal region of the
subunit (Wallner et al.,
1996
) and regulates the activity of
subunit by changing Ca2+ and voltage sensitivity and/or channel
kinetics. Although only one major type of
subunit with splice
variants has been described, several subtypes of
subunit have been
cloned and are suggested to be responsible for the differential
characteristics of BK channels in various tissues (Brenner et al.,
2000a
; Uebele et al., 2000
; Xia et al., 2000
). Synthetic openers such
as NS-1619 and BMS-204352 are activators of BK
subunit, whereas
dehydrosoyasaponin-I (Giangiacomo et al., 1998
), 17
-estradiol
(Valverde et al., 1999
), and tamoxifen (Dick et al., 2001
) act on BK
subunit. Most of these BK channel openers, including BMS-204352,
require concentrations higher than 300 nM to increase the macroscopic
BK channel current and are therefore not highly potent activators.
Terpenoids derived from natural products (i.e., dehydrosoyasaponin-I,
maxikdiol, and L-735,334) are impermeable to cell membrane and
effective only when applied to cytoplasmic side of BK channels
(Starrett et al., 1996
; Kaczorowski and Garcia, 1999
).
The present study was undertaken to identify new molecules that are
potent BK channel openers. Our goal was to find and characterize natural products and chemically modified derivatives with a novel assay
system that we developed using recombinant BK channels in human
embryonic kidney (HEK) 293 cells and voltage-sensitive dye (Yamada et
al., 2001
). Novel compounds were discovered from terpenoids, which have
chemical structures similar to that of maxikdiol, a moderate BK channel
opener, obtained from fermentation broth of an unidentified coelomycite
(Singh et al., 1994
). We found that pimaric acid (PiMA) and related
compounds are potent BK channel openers, whereas isomeric abietic acid
was not, even though it has a chemical structure very similar to that
of PiMA.
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Methods |
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Vector Constructs, Cell Culture, and Transfection.
Restriction
enzyme-digested DNA fragments of BK
(KpnI/XbaI-double digested) and BK
1
(EcoRI/XbaI-double digested) were ligated into
mammalian expression vectors, pcDNA3.1(+) and pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA), respectively, using the TaKaRa ligation kit
Ver. 1 (TaKaRa; Yamada et al., 2001
). HEK293 cells were obtained from
Health Science Research Resources Bank (HSRRB, Tokyo, Japan) and
maintained in minimum essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (JRS Biosciences, Lenexa, KS), penicillin (100 units/ml, Wako, Osaka), and streptomycin (100 µg/ml, MEIJI SEIKA, Tokyo). Stable expression of BK
and BK
was
achieved by using calcium phosphate coprecipitation transfection techniques. G418 (1 mg/ml, Invitrogen)- and G418/Zeocin (0.25 mg/ml,
Invitrogen)-resistant cells were selected as those which were
BK
-expressing and BK
1-coexpressing, respectively. Expression of BK
and BK
transcripts was confirmed by RT-PCR. It was also confirmed by RT-PCR that neither BK
nor BK
1 subunit mRNA was detected in native HEK293 cells. Transfected cell lines were maintained in minimum essential medium supplemented with 10% fetal calf serum and
G418 (0.5 mg/ml). The functional expression of BK
was verified using
inside-out patch-clamp recordings and detected in ~90% of cells
examined. In addition, the expectation of higher activity of BK
1
than that of BK
alone was confirmed in approximately 30 cells in
five separate culture dishes (Yamada et al., 2001
). Functional
expression of BK
1 was observed in approximately 80% of cells
prepared as stable HEK
1 in this study.
Solutions.
The HEPES-buffered solution for
electrophysiological recording had an ionic composition of 137 mM NaCl,
5.9 mM KCl, 2.2 mM CaCl2, 1.2 mM
MgCl2, 14 mM glucose, and 10 mM HEPES. The pH of the solution was adjusted to 7.4 with NaOH. The pipette solution contained 140 mM KCl, 1 mM MgCl2, 10 mM HEPES, 2 mM Na2ATP, and 5 mM EGTA. Adding
CaCl2 and KOH adjusted the pCa and pH of the pipette solution to 6.5 and 7.2, respectively. During recordings of
single BK channel current in the inside-out patch-clamp configuration, the pipette solution contained the HEPES-buffered solution and the
bathing solution contained 140 mM KCl, 1.2 mM
MgCl2, 14 mM glucose, 10 mM HEPES, and 5 mM EGTA.
Selected pCa of the bathing solution was obtained by adding adequate
amount of CaCl2 and the pH was adjusted to 7.2 with NaOH as described previously (Imaizumi et al., 1996
). The
perfusion solution consistently contained 0.03% dimethyl sulfoxide
(DMSO), which was used for the solvent of pimarane compounds,
regardless of the absence or presence of these compounds, in all
experiments in this study.
Electrophysiological Experiments.
Whole-cell and inside-out
patch-clamp were applied to single cells using a CEZ-2400 amplifier
(Nihon Kohden, Tokyo, Japan) and EPC-7 amplifier (List Electronik,
Darmstadt, Germany), respectively. The procedures of
electrophysiological recordings and data acquisition/analysis for
whole-cell recording have been described previously (Imaizumi et al.,
1996
). Whole-cell currents were recorded from each single cell and
leakage currents at potentials positive to
60 mV were subtracted
digitally, assuming a linear relationship between current and voltage
in the range of
90 to
60 mV. Single-channel current analyses
were done using the software PAT V7.0C, developed by Dr. J. Dempster
(University of Strathclyde, Glasgow, UK). The resistance of the pipette
was 2 to 5 M
for whole-cell and 10 to 15 M
for inside-out patch
configurations when filled with the pipette solutions. The series
resistance was partly compensated electrically under whole-cell voltage
clamp. Whole-cell and single-channel recordings were carried out at
room temperature (24 ± 1°C).
Membrane Potential Measurements by Voltage-Sensitive Fluorescent
Dye.
The measurement of membrane potential changes by
DiBAC4(3), which is a bis-barbituric acid oxonol
dye with excitation maxima at approximately 490 nm, was performed as
described previously (Yamada et al., 2001
). Before the fluorescence
measurements, cells were incubated with 50 or 100 nM
DiBAC4(3) in HEPES-buffered solution for 20 min
at room temperature. Experiments were carried out in the constant
presence of DiBAC4(3). The fluorescence emission was collected from cell clusters using a dichroic mirror (505 nm) and a
BA filter (>520 nm). Hyperpolarization results in the extrusion of the
dye from cells and a subsequent decrease in fluorescence intensity. The
decrease in fluorescence intensity by 1% corresponded to approximately
0.5-mV hyperpolarization in the membrane potential range of
20 and
70 mV (Yamada et al., 2001
). Data collection and analyses were
performed using imaging system (ARGUS-HiSCA; Hamamatsu, Hamamatsu City,
Japan). The sampling interval of DiBAC4(3) fluorescence measurements was 10 s.
Calculation of Stable Conformations of Compounds. The most stable stereochemical structures of PiMA, abietic acid, and maxikdiol were calculated by using geometry optimization with the SYBYL (molecular mechanics; Tripos, St. Louis, MO)-PM3 (semiempirical MO calculation) methods, manipulated in the computer program Spartan 4.1.2. (SGI version; Wavefunction, Inc., Irvine, CA).
Chemicals. Most of pharmacological agents were obtained from Sigma-Aldrich (St. Louis, MO). Tetraethylammonium chloride (TEA) was from Tokyo Kasei (Tokyo, Japan), Bis-(1,3-dibutylbarbituric acid)-trimethine oxonol [DiBAC4(3)] was from Molecular Probes. Inc. (Eugene, OR), and charybdotoxin was obtained from Peptide Institute Inc. (Osaka, Japan). PiMA and related natural products contained in rosin were obtained from Helix Biotech (New Westminster, BC, Canada). Methyl pimarate was prepared by methyl esterification of PiMA with ethereal diazomethane in methanol. Dihydroisopimarinol was obtained by reduction of dihydroisopimaric acid in diethyl ether with lithium aluminum hydride at reflux. Dihydropimaric acid was obtained by partial hydrogenation of PiMA over 5% Pd on carbon in methanol. The test compounds were dissolved with DMSO. The final concentration of DMSO was 0.03% or lower.
Statistics. Data are expressed as means ± S.E.M. in the text. Statistical significance between two and among multiple groups was evaluated using Student's t test and Scheffé's test after F test or one-way analysis, respectively. * and ** indicate p < 0.05 and p < 0.01, respectively.
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Results |
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Identification of BK Channel Openers Based on Natural Products
Using a Novel Assay System.
Because some terpenoids and steroids,
such as dehydrosoyasaponin-I, maxikdiol, and 17
-estradiol, exhibit
BK channel opening activity, a survey of several types of natural
terpenoids and chemically modified derivatives was undertaken to
identify a novel BK channel opener. The assay techniques that we
recently developed were applied. This is based on a decrease in
fluorescence intensity of DiBAC4(3) corresponding
to membrane hyperpolarization by BK opening, measured in HEK293 cells
in which BK
1 had been heterologously and stably expressed. The
expression of BK
1 was detected in more than 80% of cells when
examined by inside-out patch clamp (n >70; see below).
Based on their intuitive similarity of chemical structure to that of
maxikdiol, nine resin acids shown in Fig. 1 were examined in this series of
experiments. PiMA and derivatives and abietic acid are known to be
composites of pine rosin.
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1 when either 10 µM PiMA (Fig. 2A, a) or abietic acid
(Fig. 2A, b) was applied. The fluorescence intensity was markedly
reduced by application of PiMA and it recovered partially after the
addition of 5 mM tetraethylammonium (Fig. 2A, a), suggesting that the
reduction was caused by membrane hyperpolarization induced by
activation of BK channels. Application of 10 µM abietic acid either
did not change or slightly increased the fluorescence intensity (Fig.
2A, b). Effects of other compounds listed in Fig. 1 were tested in a
manner similar to that shown in Fig. 2A. Figure 2B summarizes the
results obtained from native HEK293 cells (
) and HEKBK
1 (
).
Application of 10 µM isopimaric acid, sandaracopimaric acid,
dihydropimaric acid, dihydroisopimaric acid, and dihydroisopimarinol each induced a marked decrease in fluorescence intensity in
HEKBK
1. In contrast, 10 µM methyl pimarate, abietic acid, and
sclareol induced much smaller changes in fluorescence intensity in
HEKBK
1. Isopimaric acid, methyl pimarate, dihydroisopimarinol,
abietic acid, and sclareol induced small but statistically significant changes even in native HEK293 cells, suggesting artifacts. The responses to isopimaric acid and dihydroisopimarinol in
HEK
1 were, however, much larger than those in native HEK.
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Effects of PiMA and Related Compounds on Macroscopic BK Channel
Currents.
Effects of test compounds on BK channel currents were
examined in single HEKBK
1 under whole-cell voltage clamp. The
Ca2+ concentration in the pipette solution was
fixed at pCa 6.5 using a Ca2+-EGTA buffer.
Depolarization from
60 to + 30 mV induced outward currents in both
native HEK and HEKBK
1, but the current density was much higher in
the latter (the current density at the peak: 11.0 ± 1.8 and
95.2 ± 20.9 pA/pF, n = 5 and 6, respectively,
p < 0.05). Application of PiMA in a concentration
range of 1 to 10 µM increased outward currents in a dose-dependent
fashion in HEKBK
1 (Fig. 3A) but did
not change in native HEK (data not shown). This enhancement of outward
current by PiMA could be removed completely by washout. Effects of
other test compounds were also examined in this manner. The peak
amplitude of outward current at +40 mV in the presence of 1, 3, and 10 µM resin acids was measured relative to the value taken just before
the application (Fig. 3B). PiMA, sandaracopimaric acid, isopimaric
acid, dihydroisopimaric acid, and dihydroisopimarinol showed
significant potentiating effects on outward current at concentration of
1 µM and higher (n = 4-5, p < 0.05 versus 1.0). Abietic acid, methyl pimarate, and sclareol showed no
significant effects even at 10 µM (n = 2-5),
confirming the results obtained by using
DiBAC4(3). No increases in outward current were
observed in the presence of 5 mM TEA.
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1 in the absence and
presence of 10 µM PiMA (Fig. 4). The
outward currents elicited by depolarization from
60 mV in 10 mV steps
were enhanced at potentials positive to
20 mV. The current at +40 mV
was increased from 1214 ± 153 to 2527 ± 448 pA
(n = 5, p < 0.05). The addition of 100 nM ChTX significantly reduced the outward current to levels lower than
those under the control conditions (Fig. 4, A and B). The remaining
components in the presence of 100 nM ChTX include native delayed
rectifier K+ currents and unblocked BK
1.
The current at +40 mV in the presence of 10 µM PiMA was reduced from
2527 ± 448 pA to 499 ± 90 pA (p < 0.01 versus before the application of and in the presence of 10 µM PiMA).
Cumulative addition of ChTX in the range of 3 to 100 nM reduced outward
current in a concentration dependent manner (Fig. 4C). A
concentration-response relationship for ChTX-induced block was also
obtained in the absence of PiMA; the current amplitude at + 40 mV was
reduced from 1233 ± 226 to 205 ± 46 pA by addition of 100 nM ChTX (n = 5, p < 0.01). The
normalized data were well fitted with the Hill equation modified for
the concentration-response relationship. Iblock = (1
C)/{1 + (Kd/[ChTX])nH},
where Kd is the apparent dissociation
constant of ChTX, [ChTX] is the concentration of ChTX,
nH is the Hill coefficient, and C is
the constant. The Kd obtained from the
best fitting were 7.66 ± 2.66 and 6.72 ± 2.26 nM
(n = 5, p > 0.05), in the absence and
presence of PiMA, respectively. The nH
values, 0.847 ± 0.034 and 0.982 ± 0.161 (p > 0.05), respectively, suggest one-to-one binding of ChTX to
BK
1. The C values, which may correspond to the native
K+ currents insensitive to ChTX, were 0.077 ± 0.0226 and 0.0965 ± 0.0388 (p > 0.05),
respectively. The data in Fig. 4D show that the relationship in the
presence of 10 µM PiMA was almost identical with that in its absence.
The half-maximum concentrations of ChTX for the block of BK channel
current were almost identical each other in the absence and presence of
PiMA (7.66 and 6.72 nM). These results suggest that PiMA does not
affect the binding of ChTX to BK channels.
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Effects of PiMA on Single BK
Channel Currents.
Effects of
PiMA on single BK
channel currents were examined in inside-out patch
configuration. The pCa in the bathing solution and holding potential
were 7.0 and +40 mV, respectively. The K+
concentration in the bathing and pipette solutions was 140 mM (140 mM
K+, symmetrical conditions). The unitary current
amplitude and open probability (Po) at
+40 mV was 10.1 ± 0.2 pA and 0.00362 ± 0.00099 (n = 6), respectively. Cumulative application of 0.3, 1, 3, and 10 µM PiMA increased channel opening events in a
concentration-dependent manner (Fig. 5A).
Note that PiMA was effective on BK
even when applied to cytosolic
phase, as well as when applied from outside as shown in Fig. 3. The
effect of PiMA was completely removed by washout. The
Po was measured from the event
histogram versus current amplitude (Fig. 5B). The number of channels in
a patch was determined by elevating Ca2+
concentration from pCa 7.0 to 4.0. PiMA significantly increased the
Po at concentrations of 1 µM and
higher (Fig. 5C). The relative Po was
determined by taking Po in the control
as unity: 1.66 ± 0.19, 3.92 ± 0.78, and 13.1 ± 2.3 in
the presence of 1, 3, and 10 µM PiMA, respectively (n = 6). It was confirmed that 0.03% DMSO, which was consistently added
to the perfusion solution, did not affect
Po of BK
(at +40 mV, pCa7.0;
Po = 0.0050 ± 0.0017 and
0.0036 ± 0.0010, n = 8 and 6, p > 0.05 in the absence and presence of 0.03% DMSO, respectively).
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was measured in inside-out
patch configuration at pCa 6.0 and in 140 mM K+
symmetrical conditions (Fig. 6). The
conductance was 270.1 ± 6.1 and 270.1 ± 6.0 pS in the
absence and presence of 10 µM PiMA, respectively (n = 7, p > 0.05), indicating that PiMA does not affect
BK
channel conductance.
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Vm)/S}],
where V1/2, Vm, and S are
the voltage required for half-maximum activation, membrane potential, and slope factor, respectively. Under the control conditions, the
values of V1/2 and S are 106.0 ± 5.5 mV and
13.3 ± 1.7 mV (n = 6), respectively. In the
presence of 10 µM PiMA, the Po was increased at any potentials examined and the fitted line was shifted to
negative potentials by approximately 20 mV (Fig. 7).
V1/2 and S in the presence of 10 µM PiMA are
85.6 ± 6.4 mV (p < 0.01 versus control) and
11.8 ± 1.7 mV (p > 0.05), respectively
(n = 6). The ratio of
Po in the presence and absence of 10 µM PiMA was calculated and plotted against test potentials in Fig.
7C. These results demonstrate that the lower the
Po in the control conditions, the larger the enhancement by PiMA.
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were
examined at 0 mV in 5.9/140 mM asymmetrical conditions. When
Ca2+ concentration in the bathing solution was
elevated in a range of pCa7.0 and 5.0, Po was increased in a
concentration-dependent manner (Fig. 8, A
and B). The relationship between Ca2+
concentrations and the open probability
(Po) of BK
was fitted by
Po = (1
C)/{1 + (Kd/[Ca2+])nH},
where Kd is the apparent dissociation
constant of Ca2+, [Ca2+]
is the pCa in the bathing solution, nH
is the Hill coefficient, and C is the constant. The
Kd,
nH, and C obtained from the best fitting were pCa 5.88 ± 0.03, 4.15 ± 0.42, and 0.152 ± 0.029 (n = 5), respectively, under the control
conditions. In the presence of 10 µM PiMA, the
Po was increased at any pCa examined
and the half-maximum Po was obtained
at pCa 5.98 ± 0.03 (n = 5, p < 0.05 versus the control Kd). The Hill
coefficient (3.65 ± 0.29, p > 0.05 versus
control), which indicates four Ca2+ ions to each
subunit in the tetramer, was not significantly affected by PiMA.
The value for C was 0.102 ± 0.008 (p > 0.05 versus control) and was not affected by PiMA. When the
Po in the absence of PiMA was low, the
relative increase in Po by PiMA was marked (Fig. 8C).
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Comparison of PiMA-Induced Effects on BK
and Those on
BK
1.
The results obtained using BK
clearly indicate that
PiMA enhances Ca2+ and/or voltage-sensitivity of
BK
subunit. To determine whether PiMA acts also on BK
1 subunit,
the increase in Po by PiMA in BK
1 was compared with that in BK
in inside-out patches. It has
been established that coexpression of
1 subunit with BK
increases
Ca2+ and voltage sensitivity of BK
(Wallner et
al., 1996
; Cox and Aldrich, 2000
). Accordingly, the
Po of BK
at pCa 7.0 was markedly increased by coexpression with
1 in 140 mM K+
symmetrical conditions (from 0.00023 ± 0.00014 to 0.00359 ± 0.00076 at +20 mV, n = 9 and 8, respectively,
p < 0.01 and from 0.00396 ± 0.00079 to
0.02084 ± 0.00529 at +40 mV, n = 8 and 7, respectively, p < 0.01) (Fig.
9A). The increase in
Po by PiMA depended upon the
Po under the control conditions as
shown in Fig. 6 and 7. The Po at +40
mV in BK
was close to that at + 20 mV in BK
1 (p > 0.05). Based on this observation, effects of 10 µM PiMA on BK
at + 40 mV was compared with that on BK
1 at
+20 mV (Fig. 9B). The application of PiMA increased
Po from 0.00396 ± 0.00079 to
0.03297 ± 0.00629 (n = 8, p < 0.01) in BK
and from 0.00359 ± 0.00076 to 0.03821 ± 0.01389 in BK
1 (n = 7, p < 0.05). The ratio of Po in the presence
and absence of PiMA was 10.50 ± 2.07 in BK
and near that in
BK
1 (9.68 ± 3.01, p > 0.05), suggesting that coexpression of
1 does not significantly affect the enhancement of BK
by PiMA.
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Selectivity of PiMA on BK Channel versus Small- and
Intermediate-Conductance Ca2+-Activated K+
Channels.
To examine whether the opening action of PiMA is
selective to BK channels over other Ca2+
activated K+ channels, effects of 10 µM PiMA on
membrane potential in HEK293 cells expressing rat small (rSK2) or human
intermediate conductance Ca2+-activated
K+ channels (hSK4; hIK) (Vergara et al., 1998
;
Kaczorowski and Garcia, 1999
). It has been reported that rSK2 and hIK
are similarly activated by chlorzoxazone and related compound such as
1-ethyl-2-benzimidazolinon (1-EBIO) but that they are blocked
selectively by apamin and clotrimazole, respectively (Jensen et al.,
1998
; Cao et al., 2001
). The functional expression of rat SK2 or SK4 in
each cell examined was first verified as fluorescence decrease by 15 to
30% in response to the addition of 100 µM chlorzoxazone or 30 µM
1-EBIO (data not shown). After washout of chlorzoxazone or 1-EBIO,
application of 10 µM PiMA did not change significantly the
fluorescence intensity in these SK2- or SK4-expressing cells
(p > 0.05; n = 15-20 for each). The functional expression of SK2 or SK4 verified by fluorescence decrease (> 15%) in response to SK/IK openers was observed in approximately 70 and 30% of cells, respectively. In some experiments, cells were
challenged first by 10 µM PiMA and then by 100 µM chlorzoxazone or
30 µM 1-EBIO. Application of 10 µM PiMA did not change the fluorescence intensity by more than 5% in any cells examined, regardless of SK2 or SK4 expression (data not shown). The addition of
chlorzoxazone or 1-EBIO in the presence or absence of PiMA did induce
the decrease in fluorescence intensity by 15 to 30% in cells, which
presumably expressed SK2 or SK4. The decreased fluorescence was
recovered completely or even converted to the increase by further
addition of 100 nM apamin or 1 µM clotrimazole, respectively (10-15
cells, for each of SK2 and SK4). The fluorescence intensity of
DiBAC4(3) in HEKBK
and HEKBK
1 was not
significantly affected by 100 µM 1-EBIO (n = 6-7 for each).
A Comparison of Structure of PiMA, Abietic Acid, and
Maxikdiol.
The most stable structures for each PiMA, abietic acid,
and maxikdiol were obtained by geometry optimization based on SYBYL (Molecular Mechanics) and PM3 (Semiempirical MO Calculation) methods in
the Spartan suite of programs. Comparison of structures between PiMA
and maxikdiol indicates that the overall shapes of these molecules are
similar each other (Fig. 10). When the
tricyclic frameworks were superpositioned, the hydrophobic groups
attached to C13 are located in similar spatial positions in either PiMA and maxikdiol molecules as indicated by dotted circles and a line in
the side views (Fig. 10, side view A). It can be also assumed from the
side views that the oxygen functionality of the carboxyl groups at C4
of PiMA and the tertiary alcohol at C5 of maxikdiol interact with the
same hydrogen-bonding amino acid residue in BK
protein. Another
interesting comparison can be made by assuming oxygen atom
functionality of maxikdiol and PiMA with superimposed view shown in
Fig. 10 (side view B). The alkenyl group of PiMA and methyl and alkenyl
groups of maxikdiol are assumed to occupy the same hydrophobic pocket
of the channel receptor. The structure of PiMA was also compared with
that of abietic acid. Although the shapes of these molecules are
similar (i.e., the carboxylic acid and ring structures are well
superposed), the top and side views indicate the apparent difference in
the direction and extension of the hydrophobic moieties at C13 of these
two compounds. In particular, the alkenyl group of PiMA and the methyl
groups of abietic acid are critical. Although the exact binding domain
of these compounds to BK
protein is not known at present, we note that the hydrogen-bonding functionality around C4 and the hydrophobic region around C13 are crucial for the interaction with the channel protein
subunit.
|
| |
Discussion |
|---|
|
|
|---|
The results of this study clearly demonstrate that PiMA, a common
resin acid contained in pine rosin, increases
Po of reconstituted BK channels by
changing the voltage- and/or Ca2+ dependence of
subunit without affecting the single-channel conductance. Because
PiMA and a number of other closely related compounds activated BK
channels at 1 µM but isomeric abietic acid did not, new information
regarding the molecular basis of the structure-function relationship is revealed.
BK channel
subunits encoded by KCNMA1 and
1 subunits encoded by
KCNMB1 are the combination expressed predominantly in smooth muscles
and both were cloned from smooth muscles (Garcia-Calvo et al., 1994
;
Knaus et al., 1994
). BK
, the channel forming subunit, consists of
seven transmembrane domains with a characteristic extracellular N
terminus and a long intracellular C-terminal region (Meera et al.,
1997
). The so-called Ca2+ bowl in intracellular
C-terminal domain (Schreiber and Salkoff, 1997
) and S5 transmembrane
domain (corresponding to S4 in Kv channels) are responsible for
Ca2+- and voltage-sensitivity of the
subunit,
respectively (for review, see Vergara et al., 1998
). The
1 subunit
increases Ca2+- and voltage-sensitivity of
subunit (McManus et al., 1995
; Cox and Aldrich, 2000
). Resent
publications have demonstrated that tissue-dependent diversity of BK
channel current characteristics (such as inactivation) is mainly due to
the differential expression of
subunit subfamily. Four different
subtypes have been cloned at present (Brenner et al., 2000a
; Uebele et
al., 2000
; Xia et al., 2000
). The
1 subunit is highly expressed in
smooth muscle tissues but not in brain. The knockout of the gene
encoding
1 subunit in mice results in the increased tone of arteries
and thereby contributes to systemic hypertension (Brenner et al., 2000b
). The
4 Subunit is highly expressed in brain and is
responsible for the low sensitivity of brain BK channels to IbTx (Meera
et al., 2000
).
This diversity of BK channels, as a function of their tissue-specific
distribution of
subunits, and the substantial distribution of this
channel in tissues ranging from central nervous system to vascular
smooth muscle offers important opportunities to develop new therapeutic
agents. BK channel openers have emerged as targets for drug research
and development (Starrett et al., 1996
; Lawson, 2000
). To date,
therapeutic application for BK channel openers has focused on treatment
of stroke and urinary bladder overactivity. Other aspects of
hyperactivity of smooth muscles (e.g., in the setting of asthma,
hypertension and gastric hypermolitily have also been targeted).
Benzimidazolone derivatives such as NS-4 and NS-1619, biarylureas
(NS-1608), arylpyrrole (NS-8), and indole-3-carboxylic acid esters
(CGS-7181 and CGS-7184) have been described and characterized as BK
channel openers (Coghlan et al., 2001
). BMS-204352 (developed from
benzimidazolone) has been evaluated in clinical trials for stroke
therapy, and a reduction in brain infarct size, presumably because of
its neuroprotective effects, has been detected in the rat stroke model
(Gribkoff et al., 2001
).
In addition to these synthetic compounds, many natural products have
been evaluated as BK channel openers. Terpenoids such as
dehydrosoyasaponin I (McManus et al., 1993
), maxikdiol (Singh et al.,
1994
), and L-735,334 (Lee et al., 1995
) have been identified as BK
channel openers by use of 125I-ChTX binding
assay. Despite the structural dissimilarity, these three terpenoids
share biological profiles; they can displace 125I-ChTX, but not fully, and activate BK channel
only when applied intracellularly in electrophysiological experiments.
The very low cell membrane permeability of these terpenoids limits
their utility as therapeutic and pharmacological tools. Maxikdiol is a
component derived from fermentation broth of an unidentified coelomycite. It activates BK channel in inside-out configuration at
threshold concentration of 1 µM and has a significant effect at 3 to
10 µM (Singh et al., 1994
). In this study, we chose PiMA, its
isomers, and closely related compounds for the assay of BK channel
opener, in part, because of their close structural similarity to
maxikdiol. In contrast to maxikdiol, however, PiMA activates BK
channels in HEKBK
1 when applied externally as well as when applied to `internal phase' in inside-out patches. Its potency seems
to be slightly higher than that of maxikdiol in inside-out patch
recording, because significant activation was observed at 1 µM.
Dehydrosoyasaponin-I (Giangiacomo et al., 1998
), 17
-estradiol (Valverde et al., 1999
), and tamoxifen (Dick et al., 2001
) interact with
subunits of BK channels to increase the channel activity. On
the other hand, NS-1619 (Ahring et al., 1997
), epoxyeicosatrienoic acid
(Fukao et al., 2001
), and Evans blue (Yamada et al., 2001
) act on
subunit. Our results clearly demonstrate that PiMA interacts with
subunit but may not with
1 subunit. We also found that the
concentration-response relationship of ChTX for the inhibition of
macroscopic BK
1 channel currents was not affected by the presence
of 10 µM PiMA. This finding indicates that PiMA does not affect the
ChTX binding to BK
1, although effects of PiMA on
125I-ChTX binding were not examined in this study.
An important negative result in this study is that 10 µM abietic acid
did not show BK channel opening action despite its chemical structure
similar to PiMA as the isomer. The comparison of the stable
conformations suggests that the major difference between PiMA and
abietic acid is an extension of the hydrophobic moieties at C13 (Fig.
1). Thus, only a small difference in chemical structures at C13
markedly affects the potency. It is also of interest that sandaracopimaric acid, the diastereoisomer of PiMA with respect to C13
was equipotent as PiMA. The double bond in the substituent at C13 did
not affect the potency, because dihydropimaric acid was equipotent as
PiMA. Although the substitution of carbonic acid at C4 in
dihydroisopimaric acid with alcohol (i.e., dihydroisopimarnol) did not
significantly change the potency, the lack of this moiety, such as in
sclareol or the substitution by methyl ester abolished the activity as
a BK channel opener. These results indicate that appropriate carboxylic
acid or alcohol functionality, conferred by a strong hydrogen bonding
region at C4, is essential for potent activity of these BK channel
openers. This is consistent with the slightly higher potency of PiMA
than maxikdiol, which includes "OH" group at C5 instead of C4
(Singh et al., 1994
).
Several compounds which activate BK channel, such as terpenoids, have
been discovered using 125I-ChTX binding assay
(for review, see Kaczorowski and Garcia, 1999
). One of the limitations
of this method is that the screening paradigm includes compounds, which
act only from cytoplasmic phase of the channel but may be impermeable
to cell membrane. More importantly, this method may or may not detect
indirect action of openers, which acts on a site in
subunit
apparently distinctive from 125I-ChTX binding
site in
subunit. A direct measurement of BK channel activity is
required for the high-throughput screening of putative BK channel
openers. Application of membrane potential recording techniques using
fluorescence imaging plate reader and a recombinant expression system
is preferential (Gonzalez et al., 1999
). Our previous study
demonstrated that BK channel opening action of some agents is detected
with the system (Yamada et al., 2001
). In the present study, the
membrane hyperpolarization induced by test compounds was again detected
with the same techniques. The fact that active compounds were correctly
detected demonstrates the usefulness of the assay system. The
measurements, however, can include small but significant artifacts;
isopimaric acid, methyl pimarate, dihydroisopimaric acid, abietic acid,
and sclareol changed the fluorescence intensity even in native
(untransfected) HEK cells. Slow response voltage-sensitive dyes,
including DiBAC4(3), are oxonol derivatives that
are lipophilic and negatively charged (Plasek and Sigler, 1996
).
Depolarization of cells enhances the accumulation of negatively charged
dyes from extracellular solution. It has been suggested that the
quantum yield of the dye fluorescence is increased markedly by its
binding to unidentified cytosolic proteins (Epps et al., 1994
).
Artifacts may possibly be caused by changes induced by the test
compounds in the binding of DiBAC4(3) to
cytosolic proteins.
It is clear that PiMA is specific to BK channel over SK and IK channels
as an opener and may not block them either. PiMA did not change the
membrane potential in HEK293 cells expressing SK2 or SK4, which
markedly hyperpolarizaied by SK/IK channel openers, chlorzoxazone, and
1-EBIO (Jensen et al., 1998
; Cao et al., 2001
). Genetically and even
functionally in some aspects, KCNMA (BK) is closer to voltage-dependent
K+ (Kv) channels rather than KCNN (SK and IK),
because of its voltage-sensitive domain (Vergara et al., 1998
).
Ca2+-sensing mechanisms of BK channels are also
different from SK/IK channel (Shieh et al., 2000
). It is reasonable,
therefore, that BK channel openers reported so far are selective over
SK and IK channels (Kaczorowski and Garcia, 1999
; Coghlan et al., 2001
) and is also the case for PiMA. Effects of PiMA on cloned Kv channels and other ion channels such as voltage-dependent
Ca2+ channels remain to be determined.
In conclusion, our results provide the first direct evidence that PiMA
and related compounds abundant in pine rosin have novel BK channel
opening actions. In contrast to maxikdiol, PiMA was effective from
either side of cell membrane. PiMA acts on
(but not
1) subunit
to increase Ca2+ and voltage-sensitivity of this
K+ channel complex. The fact that abietic acid,
which has close structural similarity to active pimarans, does not have
a channel opening action demonstrates that a very small and
well-defined change of the moiety, presumably at C13, markedly affects
the potency as a BK channel opener. The present results provide
important molecular information regarding the mechanisms involved in
agonist induced opening of BK
. This information is being used in our program aimed for synthesis of a novel opener.
| |
Acknowledgments |
|---|
We thank Dr. Wayne Giles (University of Calgary, Calgary, Canada) for providing data acquisition and analysis programs for macroscopic current analyses and also for critical reading of this manuscript. We also thank Dr. John Dempster (University of Strathclyde, Glasgow, UK, 1987-1994) for providing data acquisition and analysis programs for single channel analyses. C-DNA of rSK2 was supplied by Dr. H. Taniguchi (Tanabe Seiyaku Co. Ltd. Toda, Saitama, Japan), to whom we are also grateful.
| |
Footnotes |
|---|
Received February 11, 2002; Accepted July 16, 2002
This work was supported by grant-in-aid for scientific research by Japan Society for the Promotion of Sciences and also by grant-in-aid for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation (to Y.I.).
Address correspondence to: Yuji Imaizumi, Ph.D., Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan. E-mail: yimaizum{at}phar.nagoya-cu.ac.jp
| |
Abbreviations |
|---|
BK, large-conductance
Ca2+-activated K+ channel;
1-EBIO, 1-ethyl-2-benzimidazolinon;
BK
, BK channel
subunit;
BK
1, BK
channel
1 subunit;
BK
1, BK channel
plus
1 subunit;
[Ca2+]i, intracellular Ca2+
concentration;
ChTX, charybdotoxin;
DiBAC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol;
DMSO, dimetyl
sulfoxide;
HEK, human embryonic kidney;
HEKBK
, HEK293 cells
expressing BK
;
HEKBK
1, HEK293 cells expressing BK
1;
IK, intermediate Ca2+-activated K+ channel;
PiMA, pimaric acid;
Po, open probability;
RT-PCR, reverse transcription-polymerase chain reaction;
SK, small-conductance
Ca2+-activated K+ channel;
TEA, tetraethylammonium.
| |
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