The Antiarrhythmic Agent Cibenzoline Inhibits KATPChannels by Binding to Kir6.2

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

251, 477– 481 (1998)

RC989492

The Antiarrhythmic Agent Cibenzoline Inhibits KATP Channels by Binding to Kir6.2 Eri Mukai,* Hitoshi Ishida,*,† Minoru Horie,‡ Akinori Noma,§ Yutaka Seino,* and Makoto Takano§ *Department of Metabolism & Clinical Nutrition, ‡Department of Cardiovascular Medicine, and §Department of Physiology & Biophysics, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; and †Third Department of Internal Medicine, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan

Received September 13, 1998

We reported previously that cibenzoline, an antiarrhythmic agent, inhibits the ATP-sensitive K1 (KATP) channels of pancreatic b-cells through a binding site distinct from that for glibenclamide. In the present study, we have determined the locus of the action of cibenzoline on KATP channels reconstituted with mutant Kir6.2 and SUR1. We expressed a C-terminal truncated Kir6.2 (Kir6.2DC26) with and without SUR1 in COS7 cells. Both Kir6.2DC26 and Kir6.2DC26 1 SUR1 formed functional KATP channels. Glibenclamide inhibited Kir6.2DC26 1 SUR1 channels but failed to inhibit Kir6.2DC26. In contrast, cibenzoline inhibited equally Kir6.2DC26 and Kir6.2DC26 1 SUR1 channels, in a dose-dependent manner, the half-maximal concentrations of channel inhibition being 22.2 6 6.1 and 30.9 6 9.4 mM, respectively. Furthermore, we determined also that [3H]cibenzoline bound to Kir6.2DC26. These findings confirm that cibenzoline inhibits KATP channels by a novel inhibitory mechanism in which cibenzoline directly affects the pore-forming Kir6.2 subunit rather than the SUR1 subunit. © 1998 Academic Press

The ATP-sensitive K1 (KATP) channels of pancreatic b-cells play an important role in linking intracellular metabolism with membrane potential. Elevation of plasma glucose raises the intracellular ATP, inhibiting the KATP channels, depolarizing the b-cell membrane, and then activating the voltage-dependent Ca21 channels. The resultant elevation of intracellular Ca21 triggers exocytosis of the insulin granules (1). Recent molecular biological studies reveal that the pancreatic b-cell KATP channel is composed of two subunits: a sulfonylurea receptor (SUR1), a member of the ATP-binding cassette superfamily, and an inwardly rectifying K1 channel (Kir6.2), which forms the ionpore (2). Many KATP channel modulators such as nicorandil, pinacidil, and glibenclamide (3) seem to target

SUR, the regulatory subunit, but tolbutamide has been shown to act on both SUR1 and Kir6.2 (4), and phentolamine to inhibit the pore-forming subunit (5). Cibenzoline, a class Ia antiarrhythmic agent, also has been reported to induce sporadic hypoglycemia (6 – 8) by stimulating insulin secretion from the pancreatic b-cells (9). In fact, we and others have found that cibenzoline inhibits the KATP channels of pancreatic b-cells (10, 11). Furthermore, we have found evidence that the binding site of cibenzoline is distinct from that of glibenclamide (10). In the present study, we have identified the site of the action of cibenzoline in reconstituted KATP channels. Our results demonstrate that cibenzoline, unlike glibenclamide or the K1 channel openers, targets the Kir6.2 subunit rather than SUR1 subunit. MATERIALS AND METHODS Molecular biology. Kir6.2DC26, in which the last 26 amino acids were truncated from the C-terminal, was constructed by PCR, inserting a stop codon at the appropriate position. Kir6.2DC26 and SUR1 cDNA (12) were subcloned into the pCl vector which had the CMV promoter/enhancer (Promega, Madison, WI). Green fluorescent protein cDNA (GFP A65T) (13) was subcloned into the pCA vector which had the CAG promoter (14). Cell culture and transfection. COS7 cells were plated on coverslips at a density of 4 3 104 per dish (35 mm in diameter) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal calf serum. Two days later, each cocktail of pCl-SUR1 (0.4 mg), pCl-Kir6.2DC26 (0.4 mg) and pCA-GFP (0.2 mg), or of pClKir6.2DC26 (0.8 mg) and pCA-GFP (0.2 mg) was co-transfected into the COS7 cells using LipofectAMINE and Opti-MEM (GIBCO BRL, Grand Island, NY). The cells were incubated for 24 – 48 h before the electrophysiological study. Electrophysiology. Single-channel recording was performed in the inside-out configuration of the patch-clamp technique using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Coverslips were transferred to a test chamber and were perfused with bath solution containing (in mM) 135 NaCl, 5 KCl, 5 CaCl2, 2 MgSO4, and 5 Hepes (pH 7.4 with NaOH). Transfected cells could be identified by green fluorescence under a microscope. Patch pipettes (resistance 3– 6 MV) were filled with K1 external solution containing 140 KCl, 2 CaCl2, and 5 Hepes (pH 7.4 with KOH). After a giga-ohm seal

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FIG. 1. Effects of cibenzoline on the channel activities of Kir6.2DC26 plus SUR1 (A) and Kir6.2DC26 alone (B). The current traces are recorded in the inside-out configuration at the transmembrane potential of 260 mV. The dotted lines indicate the closed channel levels. The bars above current traces indicate the applications of 1 mM and 100 mM cibenzoline, 1 mM ATP, and 1 mM glibenclamide, respectively.

was established, the patch membrane was excised in a control internal solution containing 135 KCl, 10 NaOH, 0.1 CaCl2, 2 MgSO4, 1 EGTA, and 5 Hepes (pH 7.4 with KOH). K2ATP (Sigma, St. Louis, MO) was dissolved in control internal solution, and cibenzoline (Fujisawa Pharmaceutical, Osaka, Japan) and glibenclamide (Hoeschst Japan, Tokyo, Japan) were prepared respectively as 1 M and 10 mM

stock solutions in DMSO and diluted in control internal solution as required. Test solution was applied by a Y-tube apparatus which can exchange solution within 50 ms (15). All experiments were performed at room temperature (22–25°C). Current signal was filtered at 2 kHz and stored on a DAT tape recorder (TEAC, Tokyo, Japan). For computer analysis, the current signals were replayed and digi-

FIG. 2. The dose dependency of channel inhibition by cibenzoline. (A) Dose–inhibition curves of cibenzoline measured in Kir6.2DC26 (closed circles) and Kir6.2DC26 1 SUR1 (open circles). Relative amplitudes (MPC/MPCcontrol) are plotted against concentrations of cibenzoline. The curves were fitted using the Hill equation (see text). (B) Comparison of the IC50 values measured in Kir6.2DC26 (22.2 6 6.1 mM) and Kir6.2DC26 1 SUR1 (30.9 6 9.4 mM). There is no significant difference between these values (p . 0.05, Student’s unpaired t test). 478

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tized at 5 and 1 kHz through Digipack 1200A interface (Axon Instruments). Data analysis was performed using pClamp6 software (Axon Instruments) on an IBM-PC compatible computer. The unitary amplitudes of open channel current were estimated by analysis of the amplitude distribution. The integral of open channel current was divided by time for integration (mean patch current; MPC). MPCcibenzoline was measured after the current inhibition by cibenzoline reached a steady state, and it was normalized by MPCcontrol measured in the preceding control internal solution. In each set of data, we measured the half-maximal concentration for inhibition (IC50) and the Hill coeficient (nH) using the equation MPCcibenzoline /MPCcontrol 5 1/$1 1 ~@CBZ]i /IC50)nH%. In the dose–response curve, the fitted lines were drawn using the average values of IC50 and nH. Statistical data were expressed as means 6 SE. [3H]Cibenzoline binding study. The amount of specific binding of [3H]-cibenzoline was assessed using COS7 cells expressing Kir6.2DC26, as previously reported (16). Twenty-four hours after the transfection, the efficacy of transfection was verified by GFP fluorescent. All the cells were then collected by 0.25% trypsin/1 mM EDTA (GIBCO BRL). Cotransfection of pCl vector and pCA-GFP was used for the control.

RESULTS AND DISCUSSION A functional KATP channel (Kir6.2-SUR1)4 is probably a heterooctamer (17–19) because (Kir6.2)4 alone does not function as a channel and a SUR subunit is generally required for a pore-forming subunit to open. However, when the last 26 amino acids was truncated from the C-terminus of Kir6.2, Kir6.2DC26 was found to open in the absence of SUR1 (20), enabling examination of the direct effect of certain drugs on the poreforming Kir6.2 subunit. We expressed Kir6.2DC26 with and without SUR1, and compared the effects of glibenclamide and cibenzoline on channel activity. As shown in Fig. 1A, we consistently observed functional channel activity of Kir6.2DC26 1 SUR1 in the inside-out patch membrane excised from GFP-positive COS7 cells. In this condition, ATP inhibited the channels in a reversible manner, and glibenclamide also exerted an inhibitory effect on the channels. One millimolar cibenzoline inhibited the channel activity most completely, as previously reported of KATP channels in native tissue (10, 16), and the recovery of channel activity was delayed after the wash-out of cibenzoline. Figure 1B shows the channel activity of Kir6.2DC26 alone. The level of functional expression of Kir6.2DC26 was lower than that of Kir6.2DC26 1 SUR1. One hundred mM cibenzoline also inhibited channel activity. The time course of the onset and wash-out of the effect of cibenzoline was very similar to that recorded in Kir6.2DC26 1 SUR1 channels. However, glibenclamide had no effect on the activity of Kir6.2DC26. These findings clearly suggest that cibenzoline targeted Kir6.2DC26 while glibenclamide did not.

FIG. 3. The traces and amplitude histograms of Kir6.2DC26 channels in the absence (A) or presence (B) of 10 mM cibenzoline. The current traces are recorded in the inside-out configuration at the transmembrane potential of 260 mV. The dotted lines indicate the closed channel levels. The Gaussian fit to the amplitude histograms give the unitary amplitudes 4.1 pA (A) and 4.0 pA (B), respectively. Open probability was 0.501 in control and 0.357 in the presence of 10 mM cibenzoline.

This is further supported by the dose-inhibition curves for cibenzoline. In Fig. 2A, the dose–inhibition curves measured in Kir6.2DC26 (closed circle) and in Kir6.2DC26 1 SUR1 (open circle) overlapped almost completely. When we fitted the Hill equations to these data points, the half-maximal concentrations for the inhibition (IC50) were 22.2 6 6.1 and 30.9 6 9.4 mM, respectively, with no significant difference between them (p . 0.05, Fig. 2B), as was the case for the Hill coefficient (1.0 for Kir6.2DC26 and 1.1 for Kir6.2DC26 1 SUR1; p . 0.05). Thus, coexpression with SUR1 did not modulate the effect of cibenzoline, which seems intrinsic to Kir6.2. Cibenzoline did not modulate the single channel conductance of Kir6.2DC26. Figure 3 shows the amplitude histograms of Kir6.2DC26 channels measured in the

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tion of intracellular ATP, suggesting that the signal transmission between SUR1 and Kir6.2 might be functionally impaired. However, even in this condition, the efficacy of cibenzoline to inhibit KATP channel activity was not altered (16), due probably to the action site of cibenzoline being located on Kir6.2, as has been demonstrated in the present study. ACKNOWLEDGMENTS

FIG. 4. The specific binding of [3H]cibenzoline to Kir6.2DC26. The [3H]-cibenzoline binding to expressed pCl-Kir6.2DC26 is compared with that to expressed pCl vector as control. Data are means 6 SE of triplicate determinations. *Significant difference: p , 0.05.

absence and presence of 10 mM cibenzoline. The unitary amplitude was 4.1 and 4.0 pA, respectively. No apparent change was found in the single channel kinetics. We also obtained direct evidence that the cibenzoline bound to Kir6.2DC26 (Fig. 4). The specific binding of [3H]-cibenzoline was significantly higher in the COS7 cells transfected with cDNA of Kir6.2DC26 than in the control cells (p , 0.05). Cibenzoline possesses the imidazoline ring in its chemical structure. Phentolamine, an a-adrenergic receptor antagonist, also possesses the imidazoline ring. In fact, phentolamine has also been reported to inhibit KATP channels in the pancreatic b-cells and in the insulin-secreting cell line by a mechanism other than blockade of the a-adrenergic receptor (21, 22). Using C-terminal truncated Kir6.2, Proks et al. have clearly demonstrated that phentolamine directly inhibits Kir6.2 (5). Cibenzoline also inhibits Kir6.1 expressed in the NIHT3T cell line (23). Accordingly, compounds which have an imidazoline ring might generally have an inhibitory effect on Kir6.x channels. Glucose-induced insulin secretion is decreased under the condition of non-insulin-dependent diabetes mellitus (NIDDM). We (24, 25) and others (26) have shown that this is due to a defect of glucose metabolism in the pancreatic b-cells. Sulfonylureas are widely used as a potent hypoglycemic agent in therapy of NIDDM. However, they become less potent in reducing blood glucose levels during long-term treatment, along with the general deterioration of NIDDM. In this respect, we have recently reported that during sustained metabolic inhibition the potency of glibenclamide to inhibit KATP channel activity is lost concomitantly with the deple-

We are grateful to Drs. L. Aguilar-Bryan and J. Bryan for generously providing the SUR1 clone, to Dr. K. Moriyoshi for the GFP clone, and to Professor J. Miyazaki for the CAG promoter. We also thank Ms. K. Tsuji for technical support. This study was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science, and Culture; by the Committee of Experimental Models of Intractable Diseases of the Ministry of Health and Welfare of Japan; by a grant provided by the Japan Diabetes Foundation; and by a grant from the “Research for the Future” Program of the Japan Society for the Promotion of Science (JSPS-RFTF97I00201).

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