The effect of metabolic inhibition on the blocking of β-cell ATP-sensitive K+ channels (KATP channels) by glibenclamide was investigated using a patch-clamp technique. Inhibition of KATP channels by glibenclamide was attenuated in the cell-attached mode under metabolic inhibition induced by 2,4-dinitrophenol. Under a low concentration (0.1 μM) of ATP applied in the inside-out mode, KATP channel activity was not fully abolished, even when a high dose of glibenclamide was applied, in contrast to the dose-dependent and complete KATP channel inhibition under 10 μM ATP. On the other hand, cibenzoline, a class Ia antiarrhythmic agent, inhibits KATP channel activity in a dose-dependent manner and completely blocks it, even under metabolic inhibition. In sulfonylurea receptor (SUR1)- and inward rectifier K+ channel (Kir6.2)-expressed proteins, cibenzoline binds directly to Kir6.2, unlike glibenclamide. Thus, KATPchannel inhibition by glibenclamide is impaired under the condition of decreased intracellular ATP in pancreatic β-cells, probably because of a defect in signal transmission between SUR1 and Kir6.2 downstream of the site of sulfonylurea binding to SUR1.

1 We investigated the effect of cibenzoline (a class la antiarrhythmic drug) on basal insulin secretory activity of rat pancreatic islets and ATP-sensitive K+ channels (KATP) in single pancreatic cells of the same species, using radioimmunoassay and patch clamp techniques. 2 Micromolar cibenzoline had a dose-dependent insulinotrophic action with an EC50 of 94.2 + 46.4 gM. The compound inhibited the activity of the KATP channel recorded from a single fl-cell in a concentration-dependent manner. The IC50 was 0.4 gM in the inside-out mode and 5.2 jgM in the cellattached mode, at pH 7.4. 3 In the cell-attached mode, alkalinization of extracellular solution increased the inhibitory action of cibenzoline and the IC50 was reduced from 26.8 jM at pH 6.2 to 0.9 gM at pH 8.4. On the other hand, the action of cibenzoline in the excised inside-out mode was acute in onset with a small IC50, indicating that the drug attains its binding site from the cytoplasmic side of the cell membrane. 4 In the inside-out mode, micromolar ADP reactivated the cibenzoline-blocked KATP channels in a manner similar to that by which ADP restored ATP-dependent block of the channel.

Recombinant ATP‐sensitive K+ channels (KATP channels) were heterologously expressed in the NIH3T3 mouse cell line, and the electrophysiological properties were studied using patch‐clamp techniques. The NIH3T3 cell lines transfected with the inwardly rectifying K+ channel Kir6.1 alone or with both Kir6.1 and cystic fibrosis transmembrane conductance regulator (CFTR) exhibited time‐independent K+ currents with weak inward rectification. In contrast, no measurable K+ conductance was observed in mock‐transfected cells or in cells transfected with CFTR alone. Regardless of co‐transfection with Kir6.1, the transfection with CFTR produced a Cl− conductance that was activated by cell dialysis with cAMP (1 mM). The conductance was reversibly suppressed by glibenclamide (30 μM). Whole‐cell currents at +60 mV were blocked in a concentration‐dependent manner by Ba2+ ions with similar IC50 values: 89.3 ± 23.3 μM (Kir6.1 alone) and 67.3 ± 24.9 μM (Kir6.1‐CFTR). The currents recorded from Kir6.1‐transfected cells were not affected by glibenclamide, whereas glibenclamide did inhibit the conductance expressed in cells co‐transfected with CFTR (IC50= 35.9 ± 6.6 μM). In the cell‐attached mode with a 150 mM K+ pipette solution, both Kir6.1‐ and Kir6.1‐CFTR‐transfected cells displayed a class of K+ channels showing weak inward rectification and a slope conductance of 50.7 ± 1.0 and 52.4 ± 4.9 pS, respectively. In the inside‐out mode, the single‐channel currents recorded from both types of cells were not inhibited by intracellular ATP (1 mM). However, glibenclamide was found to block the single‐channel activities in the co‐transfected cells.

Several antiarrhythmic agents with Na-channel blocking action have been shown to inhibit cardiac K(ATP) channels. We used cibenzoline to examine its precise target site using patch-clamp techniques and receptor binding assays in guinea-pig ventricular myocytes. Exposure of myocytes to a glucose-free perfusate containing 1 mM cyanide produced a time-dependent shortening of the action potential duration (APD) in the current-clamp mode. Cibenzoline (30 microM) slowed the development of APD shortening (APD90 to approximately 91% vs. approximately 55% control 16 min after metabolic inhibition) at pHo 7.4, but not at pHo 6.4 (to approximately 60%). The pinacidil (30 microM)-induced K(ATP) currents were inhibited by cibenzoline in a pHo-dependent manner: the higher the pHo, the stronger the blocking effect of cibenzoline. The binding of [3H]-labeled cibenzoline was prevented by cibenzoline, but not by glibenclamide. Alkalinization produces a higher concentration of the uncharged form of cibenzoline, which can more easily permeate the cell membrane than the charged form. In NIH3T3 cells stably expressing Kir6.1, a putative pore-forming subunit of K(ATP) channel, cibenzoline but not glibenclamide inhibited the K conductance. Thus cibenzoline interacts with the channel pore-forming subunit of the K(ATP) channel (Kir6.2), but not the sulfonylurea receptor, from the cytosolic side after it permeates into the cell interior via the membrane lipid bilayer.

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