PNU-37883A inhibits vascular KATP channels (Wellman et al. 1999). It is selective for Kir6.1 over Kir6.2, and Kir6.1 may therefore form the pore of arterial KATP channels (Surah-Narwal et al. 1999; Kovalev et al. 2001). However, in some systems PNU-37883A cannot distinguish between different KATP channel subtypes (Cui et al. 2003). In this study, we have used Kir6.1/Kir6.2 chimeric proteins and current recordings to further investigate the basis of PNU-37883A inhibition of cloned KATP channels.
Chimeras between rat Kir6.1 and Kir6.2 were made by site-directed mutagenesis and subcloning. Channels were expressed following RNA injection into Xenopus oocytes. Membrane current was recorded by two-microelectrode voltage clamp of whole oocytes in a 98 mM K+ extracellular solution and at a membrane potential of -60 mV. Kir6.x constructs were co-expressed with either SUR1 or SUR2B, and currents were induced by 100 µM pinacidil (for SUR2B) or 100 µM diazoxide and 10 µM carbonyl cyanide m-chlorophenyl-hydrazone (for SUR1). Solutions and drugs were delivered by chamber perfusion (flow rate, 2 ml min-1; chamber vol., 200 µl). Kir6.1, Kir6.2, and chimeras between Kir6.1 and Kir6.2 were expressed with SUR2B, and fractional inhibition by 10 µM PNU-37883A reported.
Channels containing Kir6.1 were more sensitive to inhibition than those containing Kir6.2 (fractional inhibition: means ± S.E.M., 0.70 ± 0.05, n = 10, cf. 0.07 ± 0.03, n = 5). A chimeric channel with the Kir6.1 pore and the Kir6.2 intracellular N and C terminal domains was PNU-37883A insensitive (0.06 ± 0.07, n = 5). A chimera with the Kir6.1 C terminus and Kir6.2 N terminus and pore was inhibited (0.48 ± 0.04, n = 5). These results, and those obtained with other chimeras, suggest the C terminus is an important determinant of PNU-37883A inhibition by Kir6.1. Similar results were seen when constructs were co-expressed with SUR1. Further chimeric constructs localised PNU-37883A sensitivity to an 87 amino acid residue-long section in the Kir6.1C terminus.
Our data show structural differences between Kir6.1 and Kir6.2 are important in determining sensitivity to PNU-37883A. This compound may prove useful in probing structural and functional differences between the two channel subtypes.
Helen Kuhlman was a British Heart Foundation PhD student (FS/98047).