Different molecular sites of action for the KATP channel inhibitors, PNU-99963 and PNU-37883A

1 We investigated the mechanism of action of two novel nonsulphonylurea ATP-sensitive potassium channel (K-ATP) inhibitors, PNU-99963 and PNU-37883A, on four types of cloned K-ATP channels. 2 Whole-cell currents were recorded in asymmetrical potassium (140 mM) gradient in HEK-293 cells stably expressing Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B or Kir6.1/SUR2B. 3 PNU-99963 potently inhibited the four K-ATP channel clones. The concentration at which half-maximum current was inhibited (IC50) was 66, 41, 43 and 11 nm for Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B and Kir6.1/SUR2B, respectively. In contrast, PNU-99963 up to a concentration of 3 muM had no significant effect on current generated in HEK-293 cells by transiently expressing Kir6.2Delta26, a C-terminal truncated pore-forming subunit of Kir6.2. 4 PNU-37883A inhibited four types of K-ATP channels, but to different extents. Inhibition of the putative smooth muscle K-ATP channel types, Kir6.2/SUR2B (IC50; 15 mum) and Kir6.1/SUR213 (IC50; 6 muM), was significantly greater than inhibition of either the pancreatic beta cell or cardiac K-ATP channel clones. Moreover, PNU-37883A significantly inhibited currents generated by expressing Kir6.2Delta26 alone, with an IC50 of 5 muM, which was significantly increased to 38 muM when Kir6.2Delta26 was expressed with SUR213. 5 In conclusion, two structurally different nonsulphonylurea compounds, PNU-99963 and PNU-37883A, inhibit K-ATP channels via different mechanisms, namely through the sulphonylurea receptor (SUR) and the pore-forming subunits, respectively, although SUR213 reduced the inhibitory effect of PNU-37883A. While PNU-99963 potently inhibits all the four cloned K-ATP channels, PNU-37883A has a degree of selectivity towards both smooth muscle K-ATP channels, but could not discriminate between them.