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CELLULAR AND MOLECULAR

Molecular Mechanisms for Large Conductance Ca²⁺-Activated K⁺ Channel Activation by a Novel Opener, 12,14-Dichlorodehydroabietic Acid

Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan (K.S., S.O., K.M., Y.I.); and Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (T.N., T.O.)

Received for publication August 4, 2005
Accepted September 28, 2005.

	Abstract

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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 ( or 1) 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 BK 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 1 subunit with BK did not affect the potency of diCl-DHAA, the site of action for diCl-DHAA is suggested to be BK subunit. Moreover, kinetic analysis of single channel currents indicates that diCl-DHAA opens BK 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 BK and a useful prototype compound to develop a novel BK channel opener.

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, 17-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

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Vector Constructs, Cell Culture, and Transfection. Restriction enzyme-digested DNA fragments of BK (KpnI/XbaI-double digested) and BK1 (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 BK and BK 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 BK-expressing and BK1-coexpressing, respectively.

The cDNAs encoding voltage-dependent Ca²⁺ channel 1C subunit of the rabbit (rCaV1C) and 3 subunit of the mouse (mCaV3) 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 rCaV1C and mCaV3 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 M for whole-cell and 15 to 25 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). Single channel current analyses were done using software PAT V7.0C (developed by Dr. J. Dempster, University of Strathclyde, Glasgow, Scotland). The open probability (P_o) 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

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Effects of diCl-DHAA on Macroscopic BK Channel Currents. Effects of diCl-DHAA on BK channel currents were examined in single HEKBK1 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 HEKBK1, 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 HEKBK1 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 HEKBK1 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).

View larger version (25K): [in this window] [in a new window]   Fig. 1. Effects of diCl-DHAA on macroscopic membrane currents in HEKBK1. A and B, a single HEKBK1 was depolarized from –60 to +10 mV for 150 ms under whole-cell voltage-clamp mode. diCl-DHAA was applied in a concentration range of 0.1 and 1 µM. Original current recordings at different concentrations were superimposed and are shown in A. The peak outward current amplitude at +10 mV was measured and plotted against time in B. The original traces were obtained at the time indicated by vertical arrows in the time course. It is notable that effects of 0.1 to 1 µM diCl-DHAA were completely removed by washout. C, concentration-response relationships for diCl-DHAA. Experiments were carried out in a manner typically shown in A. Data about current density at +10 mV, which was obtained by dividing peak current amplitude with cell capacitance in each cell (pA/pF), are summarized as open columns. The relative amplitude of peak outward current at +10 mV in the presence of diCl-DHAA (IdiCl-DHAA/Icontrol) was also determined by taking the amplitude in the absence of diCl-DHAA as unity (closed columns). Means ± S.E.M. are shown by columns and vertical bars, respectively. */#, p < 0.05 and **/##, p < 0.01 versus control.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Effects of diCl-DHAA on I-V relationship in BK1. A, each single HEKBK1 was depolarized from –60 mV by 10-mV steps for 150 ms under whole-cell voltage-clamp mode. The outward currents elicited by depolarization was markedly enhanced by application of 1 µM diCl-DHAA and then reduced by the addition of 100 nM iberiotoxin. B, I-V relationships were obtained in the control (open circles), in the presence of diCl-DHAA (closed circles), and after the addition of 100 nM iberiotoxin (closed triangles) in experiments such as typically shown in A. Number of examples is seven. C, voltage dependence of the potentiation of BK channel current by diCl-DHAA was reevaluated from the data shown in B. The relative potentiation of the outward currents by 1 µM diCl-DHAA was plotted against test potentials by taking that at +30 mV as unity. *, p < 0.05 and **, p < 0.01 versus unity at +30 mV.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of diCl-DHAA on single BK channel currents recorded using an inside-out patch-clamp techniques. A, single channel currents were recorded at +40 mV in a patch from HEKBK under symmetrical 140 mM K+ conditions. The free Ca2+ in the bathing solution was adjusted to pCa = 7.0. The original current traces were recorded before and after application of 1 µM diCl-DHAA and after the washout of diCl-DHAA. A closed triangle on the left side of each trace indicates the zero current level. B, amplitude histograms in the control, in the presence of 1 µM diCl-DHAA, and after the washout were obtained from the recordings shown in A. The ordinate expresses the relative area (percentage) at the corresponding amplitude in each bin (0.2 pA). C, summarized data demonstrate the relationship between concentrations of diCl-DHAA and Po of BK. Po was calculated from the histogram shown in B as the relative time spent at open state based on the total number of BK channels in the patch, which was determined by elevating Ca2+ concentration to pCa = 3.5 (open columns). The relative Po was obtained taking the Po in the absence of diCl-DHAA as unity (closed columns). Number of examples is six. */#, p < 0.05 and **/##, p < 0.01 versus control.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Structure-activity relationships of abietic acid derivatives on Po of BK. A, chemical structures of test compounds pimaric acid, abietic acid, dehydroabietic acid, and diCl-DHAA. B, concentration-response relationships for compounds listed in A. Effects of abietic acid (open triangles), DHAA (open squares), and diCl-DHAA (closed circles) were examined in experiments identical to that shown in Fig. 3 for diCl-DHAA. The data for pimaric acid (closed squares) are taken from a previous study (Imaizumi et al., 2002). The relative Po was determined taking the Po in the absence of compounds as unity (a dotted line). Means ± S.E.M. are shown by symbols and vertical bars, respectively. Number of experiments is four to six for each compound.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effects of diCl-DHAA on single channel conductance and voltage dependence of BK. Single channel currents of BK were recorded in inside-out patch configuration at pCa = 7.0 in symmetrical 140 mM K+ conditions. Recordings were obtained at several test potentials in the range of 0 to +130 mV in the absence and presence of 10 µM diCl-DHAA. Experimental conditions, except applied potentials, are the same as those shown in Fig. 3. A, original current traces at +30, +70, and +130 mV in the absence (left) and presence of 10 µM diCl-DHAA (right). B, relationship between unitary current amplitude and test potentials was plotted in a range of 0 and +70 mV in the absence (open circles) and presence of 10 µM diCl-DHAA (closed circles) and was fitted by a linear line. The single channel conductance was determined from the slope (n = 6). C, effects of diCl-DHAA on voltage dependence of BK. The relationships between Po and test potentials were obtained in the absence (open circles) and presence of 10 µM diCl-DHAA (closed circles). Number of examples is five for each. The data were fitted using the Boltzmann equation (see "Results"). The fitted lines are based on the following values of V1/2, S, and C: 110.7 mV, 10.5 mV, and 0.15 in the absence and 72.3 mV, 12.2 mV, and 0.13 in the presence of 10 µM diCl-DHAA, respectively.

(1)

where V_1/2, V_m, 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 V_1/2 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 BK 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 P_o was increased in a concentration-dependent manner (Fig. 6, A and B). The relationship between Ca²⁺ concentration and the P_o of BK was fitted by the following equation:

(2)

where K_d 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, K_d, 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, K_d 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 subunit in the tetrameric complex of a functional BK 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 P_o in the presence of 10 µM diCl-DHAA to that of the control was plotted against [Ca²⁺]_i in Fig. 6C. The lower the P_o in the control conditions, the larger the enhancement by diCl-DHAA.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effects of diCl-DHAA on Ca2+ dependence of BK. A, single channel currents of BK were recorded in inside-out patch configuration at 0 mV in asymmetrical K+ conditions (5.9/140 mM K+) in the absence and presence of 10 µM diCl-DHAA. Recordings were obtained when [Ca2+]i was 0.1, 1, or 10 µM. B, relationships between Po and [Ca2+]i were obtained in the absence (open circles) and presence of 10 µM diCl-DHAA (closed circles). Number of examples is four for each experiment. The data were fitted with the Hill equation (see Results). The fitted lines were plotted based on the following values of Kd, m, and C: 2.06, 4.65, and 0.15µM in the absence and 1.14, 3.83, and 0.10 µM in the presence of 10 µM diCl-DHAA, respectively. C, relative Po in the presence of 10 µM diCl-DHAA versus that in the absence of 10 µM diCl-DHAA was reevaluated at various [Ca2+]i from the data shown in B. **, p < 0.01 versus relative Po at 0.1 µM Ca2+.

Effects of diCl-DHAA on the kinetic properties of BK 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 BK 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 (_Cs) and its relative magnitude (A_Cs) of the slow component (4–5 times change), whereas other parameters were moderately changed (_Os, _Cf, _Ci, and A_Of) or were not affected (_Of, A_Os, A_Cf,, and A_Ci) (n = 5).

View larger version (33K): [in this window] [in a new window]   Fig. 7. Kinetics of diCl-DHAA-induced activation of BK. A, single channel currents of BK were recorded at +50 mV in inside-out patch configuration at pCa = 6.5 in symmetrical 140 mM K+ conditions. B, dwell-time histograms of open times before (left) and after (right) application of 10 µM diCl-DHAA. The open-time histogram in the absence and presence of diCl-DHAA was fitted by a double exponential function. Of and Os represent time constants of the fast and slow components of the open times in BK kinetics, respectively. Continuous lines show the sum of individual components (dotted lines). C, dwell-time histograms of closed times before (left) and after (right) application of 10 µM diCl-DHAA. The closed time histogram was fitted by a triple exponential function. Cf, Ci, and Cs represent time constants of the fast, intermediate, and slow components of closed times, respectively.

Comparison of diCl-DHAA-Induced Effects on BK with Those on BK1. The enhancement of single BK channel activity by diCl-DHAA indicated the direct action of this compound on BK. It has been, however, established that coexpression of 1 subunit with BK increases the sensitivity of BK to Ca²⁺ and voltage (Wallner et al., 1996; Cox and Aldrich, 2000). To determine whether diCl-DHAA also acts on the functional coupling between BK and 1 subunits, the increase in P_o by diCl-DHAA in BK1 was compared with that in BK in inside-out patches. The P_o of BK at pCa = 7.0 was increased at any test potential by coexpression with 1 subunit (Fig. 8A). Since the increase in P_o by diCl-DHAA depended on a basal P_o before application (Fig. 6C), effects of diCl-DHAA on BK at +40 mV were compared with those on BK1 at +20 mV. The basal P_o 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 P_o to 0.0180 ± 0.0058 in BK (n = 5; p < 0.05) and to 0.0208 ± 0.0037 in BK1 (n = 5; p < 0.01). The ratio of P_o in BK in the presence and absence of diCl-DHAA was 7.2 ± 0.8 and therefore not significantly different from that in BK1 (11.1 ± 3.1; p > 0.05). This finding strongly suggests that coexpression of 1 subunit did not affect the diCl-DHAA-induced enhancement of BK.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Comparison of effects of diCl-DHAA on single channel currents due to BK or BK1. A, relationships between Po and test potentials were obtained in HEKBK (open squares) and HEKBK1 (closed squares). Recordings were obtained at pCa = 7.0 in symmetrical 140 mM K+ conditions. Number of examples is five for each experiment. The data were fitted using the Boltzmann equation (see Results). The fitted lines are based on the following values of V1/2, S, and C: 110.7 mV, 10.5 mV, and 0.15 in HEKBK and 96.3 mV, 11.4 mV, and 0.20 in HEKBK1, respectively. B, effect of 1 µM diCl-DHAA on Po of BK1 was compared with that of BK. The Po of BK in the absence of diCl-DHAA was 0.00382 ± 0.00104 at +40 mV and close to Po of BK1 at +20 mV (0.00346 ± 0.00079; p > 0.05). Number of examples is five for each. *, p < 0.05 and **, p < 0.01 versus the corresponding control.

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 BK were compared with those on CaV channel currents in HEK293 cells, which coexpressed 1 subunit of rabbit CaV channel and 3 subunit of mouse CaV1C3. Here, effects of pimaric acid on CaV1C3 were also examined. The inward currents through CaV1C3 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). CaV1C3 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 BK 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.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Inhibitory effects of diCl-DHAA and pimaric acid on CaV channel currents. A, representative current traces obtained from HEKs coexpressing rabbit 1C and mouse 3 with GFP (see Materials and Methods). The inward currents were elicited by 150-ms depolarizing pulses to +10 mV from a holding potential of –60 mV in the absence and presence of 0.3 or 1 µM diCl-DHAA. B, summary of effects of diCl-DHAA and pimaric acid on CaV channel currents (closed columns). For comparison, their effects on BK are also illustrated. Inhibitory effects of diCl-DHAA and pimaric acid on CaV channel currents were examined in experiments identical to that shown in A, and the relative amplitude of inward currents at +10 mV in the presence of each compound was determined taking the amplitude in the absence as unity. For revaluation of effects of diCl-DHAA and pimaric acid on BK, the same set of data shown in Fig. 3 (present study) and Fig. 5 (Imaizumi et al., 2002) were used. Means ± S.E.M. are shown by columns and vertical bars, respectively. The number in parenthesis denotes number of cells used. *, p < 0.05 and **, p < 0.01 versus unity.

	Discussion

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In the present study, we have demonstrated that diCl-DHAA is one of the most potent synthesized activators affecting the BK 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 BK 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 subunits and accessory subunits (1–4) arranged in tetramers (Vergara et al., 1998). Each subunit interacts with N-terminal region of an subunit (Wallner et al., 1996) and regulates the activity of the subunit by changing Ca²⁺ and voltage sensitivity and/or channel kinetics. Although only one major type of subunit with splice variants has been defined, the combination of the BK channel subunit encoded by KC-NMA1 and 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 BK subunit but not with the BK1 subunit (Imaizumi et al., 2002). Pimaric acid activates BK channels in HEKBK1 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 HEKBK 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), 17-estradiol (Valverde et al., 1999), and tamoxifen (Dick et al., 2001) interact with 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 BK subunit. Our results clearly showed that diCl-DHAA interacts with the BK subunit but may not interact with the BK1 subunit. We also found that 100 nM iberiotoxin completely removed the diCl-DHAA-induced potentiation of the macroscopic BK1 channel currents. This finding suggests that diCl-DHAA does not affect the iberiotoxin binding to BK1, 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 BK1 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 BK subunit to increase Ca²⁺ and voltage sensitivity. In contrast to many other BK channel openers, the effect of diCl-DHAA on BK 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 BK channel opener.

	Acknowledgements

 
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

 
doi:10.1124/jpet.105.093856.

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.

Address correspondence to: Dr. Yuji Imaizumi, 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

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