Abstract
Our recent study has revealed that 12,14-dichlorodehydroabietic acid (diCl-DHAA), which is synthetically derived from a natural product, abietic acid, is a potent opener of large conductance Ca2+-activated K+ (BK) channel. Here, we examined, by using a channel expression system in human embryonic kidney 293 cells, the mechanisms underlying the BK channel opening action of diCl-DHAA and which subunit of the BK channel (α or β1) is the site of action for diCl-DHAA. BK channel activity was significantly enhanced by diCl-DHAA at concentrations of 0.1 μM and higher in a concentration-dependent manner. diCl-DHAA enhanced the activity of BKα by increasing sensitivity to both Ca2+ and membrane potential without changing the single channel conductance. It is notable that the increase in BK channel open probability by diCl-DHAA showed significant inverse voltage dependence, i.e., larger potentiation at lower potentials. Since coexpression of β1 subunit with BKα did not affect the potency of diCl-DHAA, the site of action for diCl-DHAA is suggested to be BKα subunit. Moreover, kinetic analysis of single channel currents indicates that diCl-DHAA opens BKα mainly by decreasing the time staying in a long closed state. Although reconstituted voltage-dependent Ca2+ channel current was significantly reduced by 1 μM diCl-DHAA, BK channels were selectively activated at lower concentrations. These results indicate that diCl-DHAA is one of the most potent BK channel openers acting on BKα and a useful prototype compound to develop a novel BK channel opener.
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). BK channel activation by the Ca2+-induced Ca2+ release during excitation-contraction coupling significantly contributes to action potential repolarization/afterhyperpolarization in some smooth muscle cells (Imaizumi et al., 1998; Ohi et al., 2001).
In addition, the negative feedback control of intracellular Ca2+ concentration ([Ca2+]i) by BK channels works to protect cells from Ca2+ overload during pathophysiological conditions (Lawson, 2000). Hyperpolarization of neuronal cells by BK channel activation down-regulates the activity of voltage-dependent Na+ and Ca2+ channels and may prevent cell death, which is mainly caused by excess intracellular Ca2+ in the setting of brain ischemia following stroke. In smooth muscle cells, BK channels are also activated by spontaneous Ca2+ release from sarcoplasmic reticulum (Nelson and Quayle, 1995; Bolton and Imaizumi, 1996; Imaizumi et al., 1999) and are thought to be one of the essential regulators of resting membrane potential. Accumulated evidence indicates that the control of BK channel activity in arterial smooth muscle is one of the major determinants of vascular tone and that its abnormality can be a cause of hypertension (Brenner et al., 2000b; Wellman and Nelson, 2003; Fernández-Fernández et al., 2004).
Agents that enhance BK channel activity (BK channel openers) may therefore be effective in protecting neurons from damage following an ischemic stroke and/or in suppressing excess activity of smooth muscle tissues (Lawson, 2000). Many compounds that were found from natural products or were synthesized have been reported to be BK channel openers (Coghlan et al., 2001). Most of these BK channel openers, including BMS-204352 (Gribkoff et al., 2001), are not highly potent activators (EC50 values >300 nM) under the resting cellular conditions where intracellular Ca2+ concentration is 50 to 150 nM (Schrøder et al., 2003). Terpenoids derived from natural products— dehydrosoyasaponin-I, maxikdiol, and L-735,334— have been reported as BK channel openers (Kaczorowski and Garcia, 1999). In addition, 17β-estradiol (Valverde et al., 1999) and epoxyeicosatrienoic acids (Fukao et al., 2001) may be endogenous BK channel openers, and some transmitters and hormones can enhance BK channel activity via kinase activation (Vergara et al., 1998).
In our previous study (Imaizumi et al., 2002), novel compounds, including pimaric acid, were discovered from terpenoids, which have chemical structures similar to that of maxikdiol, a moderate BK channel opener (Singh et al., 1994). Moreover, our recent study (Ohwada et al., 2003) has revealed that chemical modification of abietic acid, an inactive compound of resin acid derivatives, to dehydroabietic acid resulted in BK channel opening, and further chemical modification to 12,14-dichlorodehydroabietic acid (diCl-DHAA) led to finding of a potent BK channel opener. However, the underlying mechanisms of diCl-DHAA-induced activation of BK channel and the selectivity against voltage-dependent Ca2+ (CaV) channel have not been defined. The present study was therefore undertaken to identify molecular mechanisms of diCl-DHAA-induced activation of BK channels and to examine the selectivity against inhibition of CaV channels by using human embryonic kidney (HEK) 293 cells as an expression system.
Materials and Methods
Vector Constructs, Cell Culture, and Transfection. Restriction enzyme-digested DNA fragments of BKα (KpnI/XbaI-double digested) and BKβ1 (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 version 1 (TaKaRa, Osaka, Japan) (Yamada et al., 2001). HEK293 cell lines were obtained from Health Science Research Resources Bank (Tokyo, Japan) and maintained in minimal essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (JRH Biosciences, Lenexa, KS), 100 units/ml penicillin (Wako Pure Chemicals, Osaka, Japan), and 100 μg/ml streptomycin (Meiji Seika, Tokyo, Japan). Stable expression of BKα and BKβ was achieved by using calcium phosphate coprecipitation transfection techniques as reported previously (Imaizumi et al., 2002). G418- and G418/zeocin-resistant cells were selected as those which were BKα-expressing and BKβ1-coexpressing, respectively.
The cDNAs encoding voltage-dependent Ca2+ channel α1C subunit of the rabbit (rCaVα1C) and β3 subunit of the mouse (mCaVβ3) were kind gifts from Dr. Veit Flockerzi (Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Hamburg, Germany) and were ligated into mammalian expression vectors pcDNA3.1(+) and pTracer(+), respectively (Murakami et al., 2003). These plasmid vectors were transfected into HEK293 cells for transient expression. The functional coexpression of rCaVα1C and mCaVβ3 was successfully determined by the appearance of the inward currents and green fluorescent protein fluorescence.
Solutions. The standard 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 for whole-cell recordings of K+ currents contained 140 mM KCl, 1 mM MgCl2, 10 mM HEPES, 2 mM Na2ATP, and 5 mM EGTA. The pCa and pH of the pipette solution were adjusted to 6.5 and 7.2 by adding CaCl2 and KOH, respectively. For recordings of single BK channel currents in the excised inside-out patch configuration, the pipette solution contained the standard HEPES-buffered solution or K+-rich HEPES-buffered solution that was prepared by replacement of 134.1 mM NaCl in the standard HEPES-buffered solution with equimolar KCl. 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. The pipette solution for whole-cell recording of Ca2+ inward currents had an ionic composition of 140 mM CsCl, 1 mM MgCl2, 10 mM HEPES, 2 mM Na2ATP, and 5 mM EGTA. The pH of the pipette solution was adjusted to 7.2 by adding CsOH.
Electrophysiological Experiments. The whole-cell and insideout patch clamps were applied to single cells using CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan) and EPC-7 amplifier (List Electronics, Darmstadt, Germany), respectively. The procedures of electrophysiological recordings and data acquisition/analysis for whole-cell recording have been described previously (Imaizumi et al., 1989). The resistance of the pipette was 1.5 to 3 MΩ for whole-cell and 15 to 25 MΩ 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). Single channel current analyses were done using software PAT V7.0C (developed by Dr. J. Dempster, University of Strathclyde, Glasgow, Scotland). The open probability (Po) was measured from the event histogram plotted against current amplitude. The number of channels in a patch was determined from recordings at pCa = 3.5, and the analyses were performed only when the number of channels in a patch was less than six.
Chemicals. Most of pharmacological agents were obtained from Sigma-Aldrich (St. Louis, MO), unless mentioned otherwise. Iberiotoxin was obtained from Peptide Institute Inc. (Osaka, Japan). Pimaric acid, abietic acid, dehydroabietic acid, and diCl-DHAA were obtained from Helix Biotech (New Westminster, BC, Canada). The test compounds were dissolved with dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was 0.03% or lower.
Statistics. Data are expressed as means ± S.E.M. Statistical significance between two groups and among multiple groups was evaluated using Student's t test and Scheffé's test after F-test or one-way analysis of variance, respectively.
Results
Effects of diCl-DHAA on Macroscopic BK Channel Currents. Effects of diCl-DHAA on BK channel currents were examined in single HEKBKαβ1 under whole-cell voltage-clamp mode. The Ca2+ concentration in the pipette solution was fixed at pCa = 6.5 using a Ca2+-EGTA buffer (see Materials and Methods). Depolarization from –60 to +10 mV induced outward currents in both native HEK and HEKBKαβ1, whereas the current density was approximately 4 times larger in the latter cells as reported previously (Imaizumi et al., 2002). Application of diCl-DHAA in a concentration range of 0.1 to 1.0 μM increased the outward currents in HEKBKαβ1 in a dose-dependent manner (Fig. 1, A and B) but not in native HEK cells (data not shown). The enhancement of the outward currents in HEKBKαβ1 was completely removed by washout of diCl-DHAA (Fig. 1B). The relationship between concentrations of diCl-DHAA and corresponding responses is summarized in Fig. 1C. The increase in outward current density by diCl-DHAA was significant at a concentration of 0.1 μM and higher. Taking the current density at +10 mV in the control as unity, the relative amplitude of peak outward currents in the presence of 0.1, 0.3, and 1 μM diCl-DHAA was also plotted against concentration (Fig. 1C).
Voltage Dependence of diCl-DHAA-Induced Enhancement of BK Channel Currents. In Fig. 2, the voltage-dependent enhancement of BK channel currents was examined by analyzing effects of diCl-DHAA on the current-voltage (I-V) relationship. HEKBKαβ1 was depolarized from a holding potential of –60 mV to test potentials in the range between –50 and +30 mV with 10-mV steps (Fig. 2A). Application of 1 μM diCl-DHAA increased the currents at any test potential. Addition of 100 nM iberiotoxin, a specific BK channel blocker, removed the enhancement of outward currents, supporting that the action of diCl-DHAA was selective to BK channel currents. Figure 2B summarizes the relationships between current density of peak outward currents and test potentials in the absence and presence of 1 μM diCl-DHAA and after addition of 100 nM iberiotoxin. The current density at +30 mV was increased from 39.13 ± 7.34 to 142.55 ± 30.81 pA/pF (n = 7; p < 0.01). In Fig. 2C, the voltage dependence of diCl-DHAA-induced enhancement of BK channel currents was determined as the relationship between the relative potentiation of outward currents and test potentials by taking the potentiation at +30 mV in the presence of 1 μM diCl-DHAA as unity. The potentiation at –20 and –10 mV was significantly greater than that at +30 mV (4.48 ± 1.15 and 3.80 ± 1.09 times at –20 and –10 mV, respectively, p < 0.01, versus unity at +30 mV).
Activation of Single BKα Channel Current by diCl-DHAA and Related Compounds. Effects of diCl-DHAA on single BKα channel currents were examined in excised inside-out patch configuration. The bathing and pipette solution contained symmetrical 140 mM K+. The free Ca2+ concentration in the bathing solution was pCa7. Under these conditions, the unitary current amplitude and open probability (Po) at +40 mV was 10.1 ± 0.2 pA and 0.0028 ± 0.0005 (n = 6), respectively. The application of 1 μM diCl-DHAA increased channel activity without change in the unitary current amplitude (10.43 ± 0.28 pA, 0.0180 ± 0.0047, n = 6; Fig. 3, A and B). It is notable that diCl-DHAA was effective on BKα even when applied to the cytosolic phase. This effect of diCl-DHAA was completely removed by the washout. The Po was significantly increased by diCl-DHAA at 0.3 μM and higher concentrations (Fig. 3C), and the relative Po determined by taking Po in the control as unity was 1.26 ± 0.13, 1.92 ± 0.19, 6.24 ± 0.67, and 14.45 ± 1.72 in the presence of 0.1, 0.3, 1.0, and 3.0 μM diCl-DHAA, respectively (n = 4–6). These results are mostly comparable with those obtained under whole-cell clamp conditions (Fig. 1C). In Fig. 4, the potency of diCl-DHAA to activate BKα was compared with that of abietic acid and dehydroabietic acid, an aromatic derivative of abietic acid. The data for pimaric acid, a potent BK channel opener, were also taken from a previous study, where the potency of pimaric acid was determined under the same experimental conditions (Imaizumi et al., 2002). Even a high concentration of abietic acid at 30 and 100 μM failed to increase the activity of BKα, whereas dehydroabietic acid at 3 and 10 μM increased activity significantly. Nevertheless, diCl-DHAA was much more potent as an activator of BKα than dehydroabietic acid and pimaric acid.
Effects of diCl-DHAA on Characteristics of Single BKα Channel Currents. In Fig. 5, effects of diCl-DHAA on characteristics of single BKα channel currents were systematically examined in the excised inside-out patch configuration. The bathing and pipette solution contained symmetrical 140 mM K+. The pCa in the bathing solution was 7. The conductance of BKα, which was determined by slope of the regression line between 0 and +70 mV, was 224.8 ± 4.1 and 224.8 ± 4.3 pS in the absence and presence of 10 μM diCl-DHAA, respectively (n = 6; p > 0.05; Fig. 5B), indicating that diCl-DHAA did not affect BKα channel conductance. The inward rectification shown at high potentials (more than +80 mV) was consistent with that reported as voltage-dependent block of BK channel by Na+ (Yellen, 1984) and was not affected by diCl-DHAA. Moreover, the Po in the absence and presence of 10 μM diCl-DHAA was calculated and plotted against test potentials in Fig. 5C. Under these conditions, the increase in Po was voltage-dependent in the range of +30 to +130 mV, and a set of data were well described by Boltzmann relationship: where V1/2, Vm, S, and C is the voltage required for half-maximum activation, membrane potential, slope factor, and constant, respectively. Application of 10 μM diCl-DHAA neither changed S nor C (S, 10.5 ± 1.5 and 12.2 ± 1.3 mV; C, 0.15 ± 0.03 and 0.13 ± 0.02 in the absence and presence of diCl-DHAA, respectively; n = 5), whereas it significantly shifted V1/2 to a more negative potential (110.7 ± 3.3 and 72.4 ± 5.7mV, respectively; n = 5; p < 0.01).
In Fig. 6, the effects of diCl-DHAA on Ca2+ sensitivity of BKα were examined at 0 mV in asymmetrical 5.9/140 mM K+ conditions. When Ca2+ concentration in the bathing solution was elevated in a pCa range between 7.0 and 5.0, the Po was increased in a concentration-dependent manner (Fig. 6, A and B). The relationship between Ca2+ concentration and the Po of BKα was fitted by the following equation: where Kd is an apparent dissociation constant of Ca2+, [Ca2+]i is the pCa in the bathing solution, m is a Hill coefficient, and C is a constant. Under the control conditions, Kd, m, and C, which were obtained from the best fitting, were pCa = 5.68 ± 0.03, 4.65 ± 0.92, and 0.152 ± 0.029, respectively (n = 4). In the presence of 10 μM diCl-DHAA, Kd was changed to pCa = 5.94 ± 0.03 (n = 4; p < 0.05), whereas the Hill coefficient, which indicates binding of one Ca2+ to each α subunit in the tetrameric complex of a functional BKα channel, was not significantly affected (3.83 ± 0.45; p > 0.05 versus control). C was 0.102 ± 0.008 and not affected by diCl-DHAA (p > 0.05 versus control). The relative Po in the presence of 10 μM diCl-DHAA to that of the control was plotted against [Ca2+]i in Fig. 6C. The lower the Po in the control conditions, the larger the enhancement by diCl-DHAA.
Effects of diCl-DHAA on the kinetic properties of BKα were examined in excised inside-out patches, which included only one channel (Fig. 7). These patches had a single channel event even when [Ca2+]i was elevated to pCa = 3.5. Figure 7A shows original current traces of BKα at pCa = 6.5 in the absence and presence of 10 μM diCl-DHAA. The data for open and closed dwell time in Fig. 7A were reconstituted as distribution histograms in Fig. 7, B and C, respectively. These histograms were well fitted by a double and triple exponential function, respectively (Fig. 7, B and C). As shown in Table 1, application of 10 μM diCl-DHAA caused a marked decrease in the mean closed time (τCs) and its relative magnitude (ACs) of the slow component (4–5 times change), whereas other parameters were moderately changed (τOs, τCf, τCi, and AOf) or were not affected (τOf, AOs, ACf,, and ACi) (n = 5).
Comparison of diCl-DHAA-Induced Effects on BKα with Those on BKαβ1. The enhancement of single BKα channel activity by diCl-DHAA indicated the direct action of this compound on BKα. It has been, however, established that coexpression of β1 subunit with BKα increases the sensitivity of BKα to Ca2+ and voltage (Wallner et al., 1996; Cox and Aldrich, 2000). To determine whether diCl-DHAA also acts on the functional coupling between BKα and β1 subunits, the increase in Po by diCl-DHAA in BKαβ1 was compared with that in BKα in inside-out patches. The Po of BKα at pCa = 7.0 was increased at any test potential by coexpression with β1 subunit (Fig. 8A). Since the increase in Po by diCl-DHAA depended on a basal Po before application (Fig. 6C), effects of diCl-DHAA on BKα at +40 mV were compared with those on BKαβ1 at +20 mV. The basal Po values were comparable with each other (0.0028 ± 0.0006 versus 0.0023 ± 0.0006; n = 5; Fig. 8B). The application of 1 μM diCl-DHAA increased the Po to 0.0180 ± 0.0058 in BKα (n = 5; p < 0.05) and to 0.0208 ± 0.0037 in BKαβ1 (n = 5; p < 0.01). The ratio of Po in BKα in the presence and absence of diCl-DHAA was 7.2 ± 0.8 and therefore not significantly different from that in BKαβ1 (11.1 ± 3.1; p > 0.05). This finding strongly suggests that coexpression of β1 subunit did not affect the diCl-DHAA-induced enhancement of BKα.
Selectivity of diCl-DHAA on BK Channel Versus Voltage-Dependent Ca2+Channel. To examine whether the action of diCl-DHAA is selective to BK channels over CaV channels, effects of 0.3 and 1 μM diCl-DHAA on BKα were compared with those on CaV channel currents in HEK293 cells, which coexpressed α1 subunit of rabbit CaV channel and β3 subunit of mouse CaVα1Cβ3. Here, effects of pimaric acid on CaVα1Cβ3 were also examined. The inward currents through CaVα1Cβ3 were elicited upon depolarization from a holding potential of –60 mV to test potentials in a range of –50 and +40 mV by 10-mV step every 10 s. The maximum amplitude was obtained at +10 mV (peak amplitude, 193 ± 54.4 pA; n = 6; Fig. 9A). CaVα1Cβ3 channel currents were not inhibited by 0.3 μM diCl-DHAA or pimaric acid, whereas they were significantly inhibited by both compounds at 1 μM (only diCl-DHAA; Fig. 9A). Data about effects of diCl-DHAA and pimaric acid on BKα are those shown in Fig. 3 and provided in a previous study (Imaizumi et al., 2002) and were obtained in inside-out patches at +40 mV and pCa = 7.0 under symmetrical 140 mM K+ conditions. Enhancement of BK channel activity by diCl-DHAA was significant at 0.3 and 1 μM, indicating that 0.3 μM diCl-DHAA is selective for the BK channel over the CaV channel.
Discussion
In the present study, we have demonstrated that diCl-DHAA is one of the most potent synthesized activators affecting the BKα subunit via changing the voltage and Ca2+ sensitivity of the channel. Chemical modification of abietic acid, an inactive compound, to dehydroabietic acid and diCl-DHAA provides a potent and selective BK channel opener. The latter has an inverse voltage dependence for BKα channel activation and is one of the most potent openers available by application from outside of the cell membrane.
BK channels consist of channel-forming α subunits and accessory β subunits (β1–β4) arranged in tetramers (Vergara et al., 1998). Each β subunit interacts with N-terminal region of an α subunit (Wallner et al., 1996) and regulates the activity of the α subunit by changing Ca2+ and voltage sensitivity and/or channel kinetics. Although only one major type of α subunit with splice variants has been defined, the combination of the BK channel α subunit encoded by KC-NMA1 and β subunits encoded by KCNMB1-4 provides the diversity of BK channels (McManus et al., 1995; Brenner et al., 2000a; Uebele et al., 2000), which offers opportunities of development of new therapeutic agents. Benzimidazolone derivatives such as biarylureas (NS-1608), NS-1619, BMS-204352 (Gribkoff et al., 2001), arylpyrrole (NS-8), and indole-3-carboxylic acid esters (CGS-7181 and CGS-7184) have been characterized as effective BK channel openers (Coghlan et al., 2001).
Natural products have also been evaluated as BK channel openers, and 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 active BK channel openers. Our pioneer work of pimarane compounds, which have a close structural similarity to maxikdiol, has revealed that pimaric acid is a potent BK channel opener that interacts with BKα subunit but not with the BKβ1 subunit (Imaizumi et al., 2002). Pimaric acid activates BK channels in HEKBKαβ1 when applied externally as well as when applied to the “internal phase” in inside-out patches. Its potency seems to be slightly higher than that of maxikdiol. An important comparative result in the previous study is that abietic acid did not show BK channel opening action despite of the fact that abietic acid is a structural isomer (C20H30O2) of pimaric acid.
Of importance is our recent finding that chemical modification of abietic acid to dehydroabietic acid as well as diCl-DHAA produced compounds active to the open BK channel (Ohwada et al., 2003). In the present study, we provided new information about mechanisms of diCl-DHAA-induced activation of BK channels. diCl-DHAA activated BK channels in HEKBKα when applied externally as well as when applied to the internal phase in inside-out patches. Its potency was obviously higher than that of pimaric acid in whole-cell recording under the same experimental conditions, since significant activation was observed at 0.1 μM diCl-DHAA (Imaizumi et al., 2002). Dehydrosoyasaponin-I (Giangiacomo et al., 1998), 17β-estradiol (Valverde et al., 1999), and tamoxifen (Dick et al., 2001) interact with β subunits of BK channels to increase the channel activity. In contrast, NS-1619 (Ahring et al., 1997), epoxyeicosatrienoic acid (Fukao et al., 2001), Evans blue (Yamada et al., 2001), and pimaric acid (Imaizumi et al., 2002) act on the BKα subunit. Our results clearly showed that diCl-DHAA interacts with the BKα subunit but may not interact with the BKβ1 subunit. We also found that 100 nM iberiotoxin completely removed the diCl-DHAA-induced potentiation of the macroscopic BKαβ1 channel currents. This finding suggests that diCl-DHAA does not affect the iberiotoxin binding to BKαβ1, although effects of diCl-DHAA on 125I-iberiotoxin binding were not examined in this study. Consistently, the concentration-response relationship of charybdtoxin for the inhibition of macroscopic BKαβ1 channel currents was not affected by the presence of 10 μM pimaric acid, which has a close structural analogy to diCl-DHAA (Imaizumi et al., 2002).
It is obvious from the present results that diCl-DHAA activates BK channels in a voltage- and Ca2+-dependent manner. It is very notable that the potentiation of BK channel activity by diCl-DHAA was significantly larger at negative potentials as well as at lower Ca2+ concentrations. In contrast, BMS-204352, a potent BK channel opener, caused activation of BK channel currents only at positive potentials (more than +30 mV; Gribkoff et al., 2001; Schrøder et al., 2003). To our knowledge, diCl-DHAA, and presumably pimaric acid as well, are the only compounds that show marked inversed voltage dependence for potentiation among various types of BK channel openers. This characteristic feature of diCl-DHAA can be considered particularly effective to prevent membrane depolarization, hyperexcitability, and/or excess Ca2+ influx to the cell and may be advantageous for clinical use. Moreover, diCl-DHAA decreased the time for the channels to stay in the prolonged closed states. It can be, therefore, suggested that this kinetic change in the presence of diCl-DHAA causes activation of BK channel. Niflumic acid opens BK channels mainly by decreasing the time in the long-closed states (Ottolia and Toro, 1994).
It is important to characterize the selectivity of diCl-DHAA to BK channels over that of other ion channels. We found that diCl-DHAA at concentrations of 0.3 μM or less increased BK channel activity without inhibiting CaV channels. Moreover, even though diCl-DHAA-induced inhibition of CaV channels at 1 μM was comparable with that by 1 μM pimaric acid (∼30% of the control), the activation of BK channel currents by diCl-DHAA was significantly greater than that by pimaric acid, suggesting that the potent activation of BK channels by diCl-DHAA provides more selectivity against CaV channels than that by pimaric acid. The selectivity of BK channel openers against CaV channels has not been well defined, but nordihydroguaiaretic acid or NS-1619-induced inhibition of CaV channel currents was comparable with or slightly more potent than the activation of BK channels, respectively (Holland et al., 1996; Yamamura et al., 1999). For development of potent and selective BK channel openers, scaffoldings of dehydroabietic acid may be useful (Ohwada et al., 2003).
Effects of diCl-DHAA on small (SK) and intermediate (IK) conductance Ca2+-activated K+ channels were not examined systematically in this study. BK channel openers reported so far, including pimaric acid, are however selective over SK and IK channels (Kaczorowski and Garcia, 1999; Coghlan et al., 2001; Imaizumi et al., 2002), and our preliminary data suggest that 1 μM diCl-DHAA did not affect the activities of SK2 and SK4 channels (K. Sakamoto, unpublished data). Genetically, and even functionally in some aspects, KCNMA (BK) is closer to voltage-dependent K+ channels than KCNN (SK and IK), because of the presence of its voltage-sensitive domain (Vergara et al., 1998). It is therefore worth examining the effects of diCl-DHAA on cloned voltage-dependent K+ channels, which remain to be determined.
In conclusion, our results provide new information of mechanisms underlying diCl-DHAA-induced activation of BK channels and the selectivity against CaV channels. diCl-DHAA is effective from either side of cell membrane and acts on BKα subunit to increase Ca2+ and voltage sensitivity. In contrast to many other BK channel openers, the effect of diCl-DHAA on BKα significantly showed inverse voltage dependence, i.e., larger potentiation at lower membrane potentials. In this respect, diCl-DHAA may be one of the most potent BK channel openers ever known to sensitize the negative feedback control of [Ca2+]i regulation via activation of BK channels, which suppress depolarization from resting membrane potential, and subsequently, membrane excitability. diCl-DHAA at low concentrations (<1 μM) shows selectivity to the BK channel over CaV channels and possesses higher selectivity to BK channels than pimaric acid. Dehydroabietic acid, including diCl-DHAA, is a new prototype scaffolding as a potent BKα channel opener.
Acknowledgments
We thank Dr. Wayne Giles (University of Calgary, Calgary, AB, 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) for providing data acquisition and analysis programs for single channel analyses.
Footnotes
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doi:10.1124/jpet.105.093856.
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ABBREVIATIONS: BK, large conductance Ca2+-activated K+; [Ca2+]i, intracellular Ca2+ concentration; BMS-204352, (3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one; L-735,334, 14-hedroxy 8-daucene-3,4-diol oleate; diCl-DHAA, 12,14-dichlorodehydroabietic acid; CaV, voltage-dependent Ca2+; HEK, human embryonic kidney; I-V, current-voltage; SK, small conductance Ca2+-activated K+; IK, intermediate conductance Ca2+-activated K+; CGS-7181, ethyl 2-hydroxy-1-[[(4-methylphenyl)amino]oxo]-6-trifluoromethyl-1H-indole-3-carboxylate; CGS-7184, ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate; NS-1608, N-(3-(trifluoromethyl)phenyl)-N′-(2-hydroxy-5-chlorophenyl)urea; NS-1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; NS-8, 2-amino-3-cyano-5-(2-fluorophenyl)-4-methylpyrrole.
- Received August 4, 2005.
- Accepted September 28, 2005.
- The American Society for Pharmacology and Experimental Therapeutics