Synthesis and characterization of a novel tritiated K-ATP channel opener with a benzopyran structure

1 The synthesis of a tritiated benzopyran-type opener of the ATP-dependent K-1 channel (K-ATP channel), [H-3]-PKF217-744 {(3S,4R)-N-[3,4-dihydro-2,2-d imetihyl-3-hyroxy-6-(2-methyl-4-pyridinyl)-2H-1-benzopyran-4-yl]-3-[2,6-H-3 ]pyridinecarboxamide} with a specific activity of 50 Ci mmol(-1) is describ ed. Binding of the ligand was studied in membranes from human embryonic kid ney cells transfected with the sulphonylurea receptor isoforms, SUR2B and S URA, respectively. 2 PKF217-744 was confirmed as being a K-ATP channel opener by its ability t o open the Kir6.1/ SUR2B channel, the recombinant form of the vascular K-AT P channel, and to inhibit binding of the pinacidil analogue, [H-3]-P1075, t o SUR2B (K-t = 26 nM). 3 The kinetics of [H-3]-PKF217-744 binding to SUR2B was described by rate c onstants of association and dissociation of 6.9 x 10(6) M-1 min(-1) and 0.0 9 min(-1), respectively. 4 Binding of [H-3]-PKF217-744 to SUR2B/2A was activated by MgATP (EC50 appr oximate to 3 muM) and inhibited (SUR2B) or enhanced (SUR2A) by MgADP. 5 Binding of [H-3]-PKF217-744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. K-t values were identical with those obtained using the opener [H-3]-P1075 as the radi oligand. 6 Glibenclamide accelerated dissociation of the SUR2B-[H-3]-PKF217-744 comp lex. 7 The data show that the affinity of [H-3]-PKF217-744 binding to SUR2B is a pproximate to 6 times lower than that of [H-3]-P1075. This is due to a surp risingly slow association rate of the benzopyran-type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological prope rties of the two opener radioligands are identical.