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

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 Ca -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 M and higher in a concentration-dependent manner. diCl-DHAA enhanced the activity of BK by increasing sensitivity to both Ca 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 Ca channel current was significantly reduced by 1 M 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. Large conductance Ca -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 Ca -induced Ca 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 Ca concentration ([Ca ]i) by BK channels works to protect cells from Ca overload during pathophysiological conditions (Lawson, 2000). Hyperpolarization of neuronal cells by BK channel activation down-regulates the activity of voltagedependent Na and Ca channels and may prevent cell death, which is mainly caused by excess intracellular Ca in the setting of brain ischemia following stroke. In smooth muscle cells, BK channels are also activated by spontaneous Ca 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 Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.093856. ABBREVIATIONS: BK, large conductance Ca -activated K ; [Ca ]i, intracellular Ca 2 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 Ca ; HEK, human embryonic kidney; I-V, current-voltage; SK, small conductance Ca -activated K ; IK, intermediate conductance Ca -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 -(2hydroxy-5-chlorophenyl)urea; NS-1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; NS-8, 2-amino3-cyano-5-(2-fluorophenyl)-4-methylpyrrole. 0022-3565/06/3161-144–153$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 93856/3068567 JPET 316:144–153, 2006 Printed in U.S.A. 144 at A PE T Jornals on A ril 3, 2017 jpet.asjournals.org D ow nladed from 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 Ca 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 (diClDHAA) led to finding of a potent BK channel opener. However, the underlying mechanisms of diCl-DHAAinduced activation of BK channel and the selectivity against voltage-dependent Ca (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 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 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 g/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). G418and G418/zeocin-resistant cells were selected as those which were BK expressing and BK 1-coexpressing, respectively. The cDNAs encoding voltage-dependent Ca channel 1C subunit of the rabbit (rCaV 1C) and 3 subunit of the mouse (mCaV 3) 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 rCaV 1C and mCaV 3 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 Ca 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