ATP-sensitive K؉ (K ATP ) channels are the target of a number of pharmacological agents, blockers like hypoglycemic sulfonylureas and openers like the hypotensive cromakalim and diazoxide. These agents act on the channel regulatory subunit, the sulfonylurea receptor (SUR), which is an ABC protein with homologies to P-glycoprotein (P-gp). P-gp is a multidrug transporter expressed in tumor cells and in some healthy tissues. Because these two ABC proteins both exhibit multispecific recognition properties, we have tested whether SUR ligands could be substrates of P-gp. Interaction with P-gp was assayed by monitoring ATPase activity of P-gp-enriched vesicles. The blockers glibenclamide, tolbutamide, and meglitinide increased ATPase activity, with a rank order of potencies that correlated with their capacity to block K ATP channels. P-gp ATPase activity was also increased by the openers SR47063 (a cromakalim analog), P1075 (a pinacidil analog), and diazoxide. Thus, these molecules bind to P-gp (although with lower affinities than for SUR) and are possibly transported by P-gp. Competition experiments among these molecules as well as with typical P-gp substrates revealed a structural similarity between drug binding domains in the two proteins. To rationalize the observed data, we addressed the molecular features of these proteins and compared structural models, computerized by homology from the recently solved structures of murine P-gp and bacterial ABC transporters MsbA and Sav1866. Considering the various residues experimentally assigned to be involved in drug binding, we uncovered several hot spots, which organized spatially in two main binding domains, selective for SR47063 and for glibenclamide, in matching regions of both P-gp and SUR.ABC 8 proteins form a large superfamily of mostly membrane proteins. They present as common characteristics a nucleotide binding domain able to hydrolyze ATP, which includes the sequence motifs Walker A and B and the signature C (1). As membrane active transporters, they are expressed in virtually all branches of the living reign, and they exhibit a very broad diversity of transported substrates, handled in either the direction of influx or efflux (2). In higher mammals, they are involved in various pathophysiological situations and genetic diseases (3).The K ATP channel results from the constitutive association of four pore-forming Kir6.x subunits and four regulatory SUR subunits (4, 5). In various combinations of the SUR isoforms, SUR1, SUR2A, and SUR2B, and the Kir6 isoforms, Kir6.1 and Kir6.2, these channels are present in most excitable cells, including neuronal, cardiac muscle, smooth muscle, and endocrine cells (6), where they serve to couple membrane electrical properties to intracellular metabolism.SUR, a member of the ABCC/MRP (multidrug resistanceassociated protein) subfamily of ABC proteins, is the site of action of numerous drugs that cause either closing (K ATP channel blockers) or opening (K ATP channel openers) of the Kir6.x potassium pore (7). Blockers include antidi...