Potent Activation of Large-Conductance Ca -Activated K Channels by the Diphenylurea 1,3-Bis-[2-hydroxy-5- (trifluoromethyl)phenyl]urea (NS1643) in Pituitary Tumor (GH3) Cells

1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) is reported to be an activator of human ether-à-go-go-related gene current. However, it remains unknown whether it has any effects on other types of ion channels. The effects of NS1643 on ion currents and membrane potential were investigated in this study. NS1643 stimulated Ca -activated K current [IK(Ca)] in a concentration-dependent manner with an EC50 value of 1.8 M in pituitary tumor (GH3) cells. In inside-out recordings, this compound applied to the intracellular side of the detached channels stimulated large-conductance Ca -activated K (BKCa) channels with no change in single-channel conductance. It shifted the activation curve of BKCa channels to less depolarized voltages without altering the gating charge of the channels. NS1643-stimulated channel activity depended on intracellular Ca , and mean closed time during exposure to NS1643 was reduced. NS1643 (3 M) had little or no effect on peak amplitude of ether-à-go-go-related gene-mediated K current evoked by membrane hyperpolarization, although it increased the amplitude of late-sustained components of K inward current, which was suppressed by paxilline but not by azimilide. NS1643 (3 M) had no effect on L-type Ca current. This compound reduced repetitive firing of action potentials, and further application of paxilline attenuated its decrease in firing rate. In addition, NS1643 enhanced BKCa-channel activity in human embryonic kidney 293T cells expressing -hSlo. In summary, we clearly show that NS1643 interacts directly with the BKCa channel to increase the amplitude of IK(Ca) in pituitary tumor (GH3) cells. The -subunit of the channel may be a target for the action of this small compound. NS1643 (Fig. 1) is a novel small-molecule compound that was reported to activate HERG K channels expressed in Xenopus laevis oocytes and in mammalian HEK293 cells (Casis et al., 2006; Diness et al., 2006; Hansen et al., 2006; Lu et al., 2008). This compound has also been shown to affect spontaneous rhythmic activity in the epididymal duct via the activation of erg current (Mewe et al., 2008). Earlier studies have demonstrated that NS1608, NS1619, and NS11021 (Fig. 1), which are chemical structurally related compounds of NS1643, can modulate the activity of BKCa channels (Holland et al., 1996; Strøbaek et al., 1996; Bentzen et al., 2007). Whether NS1643 can affect other types of ion channels has not been clearly investigated, although its activation of hKCNQ1 channels was shown (Diness et al., 2006). The BKCa channel, which is the product of a nearly ubiquitous, alternatively spliced gene (KCa1.1, Slo1, or KCNMA1), represents a functional subtype of K channels, which play important roles in the regulation of neuronal excitability and hormonal secretion (Ghatta et al., 2006). The work in this laboratory was partly supported by grants from the National Science Council (NSC-95-2745-B-006-002-MY2) and the Program for Promoting Academic Excellence and Developing World Class Research Centers, Ministry of Education, Taiwan. H.P., M.L. and Y.W. were partially supported by Graduate Scholarships from National Cheng Kung University Medical College. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.049106. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: AP, action potential; BKCa, large-conductance Ca 2 -activated K ; erg, ether-à-go-go related gene; HEK, human embryonic kidney; HERG, human ether-à-go-go-related gene; hSlo, human slowpoke; IK(Ca), Ca 2 -activated K current; IK(erg), ether-à-go-go related gene-mediated K current; NS1608, N-(3-(trifluoromethyl)phenyl)-N -(2-hydroxy-5-chlorophenyl)urea; PEI, polyethylenimine. 0026-895X/08/7406-1696–1704$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 6 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 49106/3411779 Mol Pharmacol 74:1696–1704, 2008 Printed in U.S.A. 1696 http://molpharm.aspetjournals.org/content/suppl/2008/09/22/mol.108.049106.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 3, 2017 m oharm .aspeurnals.org D ow nladed from This channel is believed to share many of the common structural features of homotetrameric voltage-gated K channels, including an ion-selective pore formed by transmembrane segments S5 and S6 and a voltage-sensing module formed by transmembrane segments S1 to S4. The channel is unique in that it can be dually activated by membrane depolarization and elevations in intracellular Ca concentrations. Organic compounds with varying structures capable of activating BKCa channels have been reported previously (Calderone, 2002; Wu et al., 2006; Nardi and Olesen, 2008). Because of their unique high conductance ( 200 pS), even at low probability of opening, sufficient current could flow, thereby altering the membrane potential of excitable cells. On the other hand, the modulators of BKCa channels represent a potentially therapeutic approach to a wide variety of diseases (Nardi and Olesen, 2008). In this study, we found that NS1643 can be effective in activating BKCa channels in pituitary tumor (GH3) cells and in HEK293T cells expressing -hSlo. The presence of this compound caused a shift in the voltage-dependence of BKCa channels to less depolarized voltages accompanied by a decrease in the mean closed time of the channel. Our results clearly show that in addition to stimulation of the HERG channel, NS1643 is a potent BKCa channel activator. Materials and Methods Cell Culture. GH3, a clonal cell line derived from a rat prolactin-secreting pituitary tumor, was obtained from the Bioresources Collection and Research Center (Hsinchu, Taiwan), and the detailed methodology has been described previously (Wu et al., 2000). In brief, cells were cultured in Ham’s F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 15% (v/v) heat-inactivated horse serum, 2.5% (v/v) fetal calf serum, and 2 mM L-glutamine (Invitrogen) in a humidified environment of 5% CO2/95% air. The culture medium was changed every 2 to 3 days, and cells were passaged when they reached confluence. Cell viability was often evaluated using WST-1 assay (Roche Diagnostics, Taipei, Taiwan). To promote cell differentiation, GH3 cells were transferred to a serum-free, Ca -free medium. Under these conditions, cells remained 80 to 90% viable for at least 2 weeks. The experiments were performed 5 or 6 days after cells had been cultured (60–80% conflu-