Potentiation of Large Conductance Kca Channels by Niflumic, Flufenamic, and Mefenamic Acids

2272 Biophysical Journal Volume 67 December 1994 2272-2279 Potentiation of Large Conductance KCa Channels by Niflumic, Flufenamic, and Mefenamic Acids M. Ottolia and L. Toro Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030 USA ABSTRACT Large conductance calcium-activated K+ (Kca) channels are rapidly activated by niflumic acid dose- dependently and reversibly. External niflumic acid was about 5 times more potent than internal niflumic acid, and its action was characterized by an increase in the channel affinity for [Ca2l], a parallel left shift of the voltage-activation curve, and a decrease of the channel long-closed states. Niflumic acid applied from the external side did not interfere with channel block by charybdotoxin, suggesting that its site of action is not at or near the charybdotoxin receptor. Accordingly, partial tetraethylammonium blockade did not interfere with channel activation by niflumic acid. Flufenamic acid and mefenamic acid also stimulated KCa channel activity and, as niflumic acid, they were more potent from the external than from the internal side. Fenamates applied from the external side displayed the following potency sequence: flufenamic acid niflumic acid >> mefenamic acid. These results indicate that KCa channels possess at least one fenamatereceptor whose occupancy leads to channel opening. INTRODUCTION Large conductance calcium-activated K channels are present quently purified in a sucrose gradient. Membranes obtained from the of cell types (Latorre et al., 1989; McManus, 1991). 20%:25% and 25%:30% (w/w) sucrose interface were used. Lipid bi- in a variety layers were cast from a phospholipid solution in n-decane containing a In neurons they may regulate cell firing (Gola and Crest, 1993), 5:2:3 mixture of phosphatidylethanolamine/phosphatidylserine/phos- and in smooth muscle they seem to play an important role in phatidylcholine (25 mg/ml). The voltage control side was the cis cham- maintaining visceral and vascular tone (Brayden and Nelson, ber, and the trans chamber was referred to ground. Membrane vesicles 1992; Suarez-Kurtz et al., 1991; Anwer et al., 1993; Khan et al., were applied on top of the preformed bilayer from the cis side. The activate this type of channels from the laterality of channel incorporation was determined by the voltage de- 1993). Thus, drugs that pendence of channel gating. external side or internal side (McManus et al., 1993) should be The effect of niflumic acid was tested on reconstituted KCa channels valuable pharmacological tools to modify cellular excitability, as from coronary smooth muscle with low open probability ('0.25). The well as to unveil mechanisms of Kca channel function. experimental solutions were, for the cis chamber (mM): 250 KCl, 10 Niflumic and flufenamic acids are nonsteroidal anti- MOPS, 1 HEDTA, 0.67 CaCl2 (pH 7.2, pCa 5.17); for the trans chamber compounds (Hoffmann and Faure, 1966; (mM): 5 KCI, 245 NaCl, 10 MOPS, 1 HEDTA, 0.67 CaCl2 (pH 7.20, inflammatory aromatic pCa 5.17). Variations to these solutions are indicated in the figure leg- Kohler et al., 1992) known to inhibit Cl- conductances (White ends. Calcium-activation curves were constructed by perfusing solu- and Aylwin, 1990; Korn et al., 1991; McCarty et al., 1993) and tions with different [Ca2+]i, calculated according to Fabiato (1988). A nonselective cationic channels (Gogelein et al., 1990). Recently, rapid perfusion system for a bilayer setup ('15-30 s) was used to a calcium-independent K current from jejunum smooth muscle exchange solutions. has been shown to be increased by fe- Data were acquired on line at 1 ms/point and filtered at 500 Hz using and corneal epithelium an 8-pole Bessel filter. Analysis was performed using TRANSIT (A. M. namates (Rae and Farrugia, 1992; Farrugia et al., 1993a, b). We J. VanDongen, Duke University, Chapel Hill, NC). Open probability now demonstrate that flufenamic niflumic>> mefenamic ac- was obtained from the ratio between the open time and the total time. ids can activate large conductance K,> channels from the ex- Kinetic analysis was performed in bilayers with a single channel. manner. Part ofthis work has A critical closed time of 1 s was used to obtain charybdotoxin- ternal side in a rapid and reversible ± in abstract form et al., 1993). induced blocked and unblocked times. Values are means SEM. A been presented (Foro one-tailed Student's t-test, or ANOVA and multiple comparison Tukey tests were applied; values were considered significantly different at a level p ' 0.05. MATERIALS AND METHODS Niflumic acid, mefenamic acid, and flufenamic acids were purchased Chemical Co. (St. Louis, MO). Concentrated stock solutions Coronary smooth muscle membrane vesicles were obtained as described from Sigma mM) of niflumic and flufenamic acids were done in ethanol, whereas in Toro et al. (1991). Briefly, plasma membrane vesicles from pig coro- (100 acid stock solution (100 mM) was prepared in 1:1 ethanol:dim- smooth muscle were prepared from 20 or 30 arteries. Microsomes mefenamic nary Final dilutions had a pH of 7.20 and contained at were obtained in the presence of proteases inhibitors and were subse- ethylsulfoxide (DMSO). most 1% ethanol or 0.05% DMSO. These concentrations of solvent did not increase by themselves the channel open probability. Receivedforpublication 22 February 1994 and infinalform 26 May 1994. RESULTS AND DISCUSSION Address reprint requests to Ligia Toro, Ph.D., Dept. Anesthesiology, UCLA School of Medicine, Rm. BH612 CHS, 10833 Le Conte Ave. Los Angeles, Niflumic acid activates KCa channels from the CA 90024. external side in a dose-dependent, fast, Authors' present address: Dept. Anesthesiology, University of California at and reversible manner Los Angeles, Los Angeles, CA 90024. X) 1994 by the Biophysical Society The action of niflumic acid on KCa channels from coronary 0006-3495/94/12/2272/08 $2.00 smooth muscle reconstituted into lipid bilayers was explored. Ottolia and Toro Fenamates as Kc, Channel Agonists 2273 Micromolar concentrations of niflumic acid from the exter- illustrates the activity of a channel under control conditions nal side caused an increase in channel open probability (P.) ([Ca2]i = 7 ,M), after diminution of channel activity by (60 out of 64 studied channels). For example, in 10 experi- lowering [Ca2"]i to 3 ,uM with a calcium chelator (HEDTA), ments with 100 ,uM niflumic acid channel P. increased from and its fast increase after external perfusion of niflumic acid. 0.09 ± 0.01 to 0.35 ± 0.05. When dose-response curves were The lower panel illustrates in another experiment that ni- constructed, the Kd was 261 ± 73 ,uM (n = 5) and the Hill flumic acid-induced increase ofKc. channel activity could be coefficient approximated 1 (0.99 ± 0.12). Fig. 1 A exem- readily reversed after washing out the drug. It seems, there- plifies a channel with a Kd of 132 ,uM and a Hill coefficient fore, that Kc. channels possess a specific niflumic acid re- of 0.6. A Hill coefficient near one indicates that the poten- ceptor. Furthermore, it is very likely that this receptor is tiation of Kca channels by niflumic acid involves a bimo- located in the channel protein and not in a closely asso- lecular interaction between the drug and its binding site. The ciated molecule because we have observed that external stimulatory effect of externally applied niflumic acid was niflumic is also able to stimulate a cloned KCa channel also observed in Kc. channels from other sources like uterine reconstituted in lipid bilayers (Perez et al., 1994; our un- and tracheal smooth muscle, slo Kca channel expressed in published observations). oocytes and from skeletal muscle. For example, 100 ,uM niflumic acid caused an increase in channel PO from the first three sources from 0.37 to 0.71, from 0.3 to 0.8, and from Mechanism of niflumic acid activation 0.21 to 0.55, respectively. of Kca channels Niflumic acid action took place within the time resolution Because KCa channels are voltage- and calcium-sensitive, we of our perfusion system (15-30 s), and its effect was readily decided to study whether niflumic acid affected these chan- washed out (P.-.ntro = 0.05 ± 0.01; Po-niflumic acid = 032 + nel properties. We found that niflumic acid from the external 0.07; Pw,,ashout = 0.047 ± 0.01; n = 3) even if a high dose side exerted its effect on Kc. channels by left-shifting both of niflumic acid was used. Fig. 1 B shows the time course of their voltage- and calcium-activation curves without major two such experiments where 1 mM niflumic acid was per- changes in the slopes of the curves. Fig. 2 A shows that fused to the external side of the channel. The upper graph perfusion of 100 ,uM niflumic acid to the external side in- A B Control HEDTA Niflumic Ac. Control Intemal Extemal 4- *- J , 15 pA Niflumic acid 1.0 Ts lW,UM~~~~~~~~~~~~~~~~~~~' 0.8 0.6 I1 500,uM 0.4 0.2 0.0 500 ms 0 50 100 150 200 300 Time, s 1.0 I 0.8 Control lmM Washout 0.6 +1M~m I5p1 .0 0 1.0 - 0.4 5 0.8- .0 0.6- 0.2 t 0.4- 0.0 -2 10 1 10 10 104 o 50 15DU AU 300 350 [Niflumic acid], #M Time, s FIGURE 1 Niflumic acid activates Kc channels. The action of external niflumic acid on Ca2+-activated K+ (KCa) channels was assessed using pig coronary smooth muscle plasma membranes incorporated into lipid bilayers. (A) Records illustrating Kc. channel activation by niflumic acid at different [niflumic acid], and corresponding dose-response curve. Experimental data were fitted to: Normalized PO = (1 - A)/(1 + (K112/[niflumic acid])N) + A, where K,12N = Kd and A is the channel P.

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