ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

Insulin secretagogues (sulfonylureas and glinides) increase insulin secretion by closing the ATP-sensitive K ؉ channel (K ATP channel) in the pancreatic ␤-cell membrane. K ATP channels subserve important functions also in the heart. First, K ATP channels in coronary myocytes contribute to the control of coronary blood flow at rest and in hypoxia. Second, K ATP channels in the sarcolemma of cardiomyocytes (sarcK ATP channels) are required for adaptation of the heart to stress. In addition, the opening of sarcK ATP channels and of K ATP channels in the inner membrane of mitochondria (mitoK ATP channels) plays a central role in ischemic preconditioning. Opening of sarcK ATP channels also underlies the STsegment elevation of the electrocardiogram, the primary diagnostic tool for initiation of lysis therapy in acute myocardial infarction. Therefore, inhibition of cardiovascular K ATP channels by insulin secretagogues is considered to increase cardiovascular risk. Electrophysiological experiments have shown that the secretagogues differ in their selectivity for the pancreatic over the cardiovascular K ATP channels, being either highly selective (ϳ1,000؋; short sulfonylureas such as nateglinide and mitiglinide), moderately selective (10 -20؋; long sulfonylureas such as glibenclamide [glyburide]), or essentially nonselective (<2؋; repaglinide). New binding studies presented here give broadly similar results. In clinical studies, these differences are not yet taken into account. The hypothesis that the in vitro selectivity of the insulin secretagogues is of importance for the cardiovascular outcome of diabetic patients with coronary artery disease needs to be tested. Diabetes 53 (Suppl. 3):S156 -S164, 2004 I nsulin secretagogues are widely prescribed in the treatment of type 2 diabetes. They close the ATPsensitive K ϩ channel (K ATP channel) in the membrane of the pancreatic ␤-cell, thereby depolarizing the cell and triggering insulin secretion. K ATP channels are gated by intracellular nucleotides with ATP inducing channel closure and MgADP channel opening. The ␤-cell is special in that physiological changes in plasma glucose change the intracellular ATP and ADP concentrations such that the channel opens and closes; hence, the channel functions as the glucose sensor in this cell (1-3). K ATP channel subtypes are found in many cell types. The generation of mice in which the genes for the K ATP channel subunits were deleted have shed new light on the diverse functions of the K ATP channels in various tissues in physiological and pathophysiological conditions (1). In brain, K ATP channels are involved in actions as diverse as the control of glucose homeostasis and the regulation of neuronal excitability in hypoxia (1); however, the insulin secretagogues do not cross the blood-brain barrier easily enough to affect these channels at therapeutic plasma levels (4). In several vascular beds, the K ATP channel in the vascular myocytes is involved in the regulation of vessel tone; opening is triggered in particular by stimuli inc...

ATP-sensitive K ϩ channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K ϩ channel openers. These channels are octamers of Kir6.x and sulfonylurea receptor (SUR) subunits with 4:4 stoichiometry. The properties of the opener-sensitive K ϩ channel in the vasculature are well matched by the SUR2B/Kir6.1 channel; however, the GBC sensitivity of the recombinant channel is unknown. In binding experiments we have determined the affinity of GBC for SUR2B and the SUR2B/Kir6.1 channel and compared the results with the channel blocking potency of GBC. All experiments were performed in whole transfected human embryonic kidney cells at 37°C. The equilibrium dissociation constants (K D ) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the K D value of the opener P1075 (N-cyano-NЈ-(1,1-dimethylpropyl)-NЈЈ-3-pyridylguanidine) (Ϸ5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC 50 Ϸ 43 nM. The data show that, in the intact cell: 1) SUR2B, previously considered to be a low-affinity SUR, has a rather high affinity for GBC; 2) coexpression with the inward rectifier Kir6.1 increases the affinity of SUR2B for GBC; 3) the recombinant channel exhibits the same GBC affinity as the opener-sensitive K ϩ channel in vascular tissue; and 4) the K D value of GBC binding to the octameric channel is 7 times lower than the IC 50 value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.

Aims/hypothesis Sulfonylureas and glinides close beta cell ATP-sensitive K + (K ATP ) channels to increase insulin release; the concomitant closure of cardiovascular K ATP channels, however, leads to complications in patients with cardiac ischaemia. The insulinotrope repaglinide is successful in therapy, but has been reported to inhibit the recombinant K ATP channels of beta cells, cardiocytes and non-vascular smooth muscle cells with similar potencies, suggesting that the (patho-)physiological role of the cardiovascular K ATP channels may be overstated. We therefore re-examined repaglinide's potency at and affinity for the recombinant pancreatic, myocardial and vascular K ATP channels in comparison with glibenclamide. Results Repaglinide and glibenclamide, respectively, were ≥30 and ≥1,000 times more potent in closing the pancreatic than the cardiovascular channels and they did not lead to complete inhibition of the myocardial channel. Binding assays showed that the selectivity of glibenclamide was essentially based on high affinity for the pancreatic SUR, whereas binding of repaglinide to the SUR subtypes was rather non-selective. After coexpression with Kir6.x to form the assembled channels, however, the affinity of the pancreatic channel for repaglinide was increased 130-fold, an effect much larger than with the cardiovascular channels. This selective effect of coexpression depended on the piperidino substituent in repaglinide. Conclusions/interpretation Repaglinide and glibenclamide show higher potency and efficacy in inhibiting the pancreatic than the cardiovascular K ATP channels, thus supporting their clinical use.