Identification of the potassium channel opener site on sulfonylurea receptors.

Diversity of sulfonylurea receptor (SUR) subunits underlies tissue specific pharmacology of K(ATP) channels, which represent critical regulators of electrical activity in numerous cells. Notably, the neuronal/pancreatic beta-cell receptor, SUR1, imparts high sensitivity to hypoglycemic sulfonylureas (SUs; e.g. glibenclamide) and low to potassium channel openers (KCOs; e.g. P1075), whereas the opposite drug sensitivities are conferred by cardiovascular receptors, SUR2A and SUR2B. By exchanging domains between SUR1 and SUR2B, we identify two regions (KCO I: Thr(1059)-Leu(1087) and KCO II: Arg(1218)-Asn(1320); rat SUR2 numbering) within the second set of transmembrane domains (TMDII) as critical for KCO binding. Swapping both regions reconstitutes KCO affinities and sensitivities of the donor SUR isoform. High glibenclamide affinity of SUR1 is not reduced by transfer of KCO I plus II from SUR2B, demonstrating that high SU and KCO affinity can coexist in the same SUR molecule. Consistently, high SU affinity was imparted on SUR2B by substituting the region separating KCO I and II (Ile(1088)-Val(1217)) with the corresponding domain of SUR1. We infer the receptor sites for KCOs and SUs to be closely associated within a regulatory domain (Thr(1059)-Asn(1320)) in TMDII of SURs.     

Potassium channel openers require ATP to bind to and act through sulfonylurea receptors.

KATP channels are composed of a small inwardly rectifying K+ channel subunit, either KIR6.1 or KIR6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP-binding cassette superfamily. SUR1/KIR6.2 reconstitute the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular-smooth-muscle-type KATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUR2A and SUR2B requires ATP. Non-hydrolysable ATP-analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide-binding folds (NBFs) abolish or weaken [3H]P1075 binding to SUR2B, rendering reconstituted SUR2B/KIR6.2 channels insensitive towards KCOs. The C-terminus of SUR affects KCO affinity with SUR2B approximately SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [3H]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5-8-fold higher than their potencies for activation of SUR2B/KIR6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.     

Characterization of sulfonylurea receptors in isolated human pancreatic islets

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [³H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a K_d value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182–188, 1998. © 1998 Wiley-Liss, Inc.     

Pancreatic polypeptide receptors: affinity, sodium sensitivity and stability of agonist binding

Cloned rat, human and guinea-pig Y4 pancreatic polypeptide (PP) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the rabbit Y4-like PP receptor, show a selective sensitivity to Na+ over K+ ion in PP attachment, but little sensitivity to Na+ in dissociation of bound PP peptides. Agonist binding to Y4 receptors of intact CHO cells also shows much greater sensitivity to Na+ over K+, and a tenacious attachment of the bound agonist. Binding sensitivity to K+ is greatly enhanced upon receptor solubilization. Pancreatic polypeptide sites also show large sensitivity to modulators of Na+ transport such as N5-substituted amilorides and to RFamides, as different from Y1 or Y2 receptors. Thus, PP binding is modulated by cation-induced changes in site environment (with selectivity for Na+) and ultimately results in a blocking attachment. This would support receptor operation in the presence of ion gradients, as well as prolonged agonist-delimited signaling activity (which can include partial antagonism). Also, this could point to an evolutionary adaptation enabling small numbers of PP receptors to perform extensive metabolic tasks in response to low agonist signals.     

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

Co-expression of sulfonylurea receptors and KATP channels in hamster insulinoma tumor (HIT) cells. Evidence for direct association of the receptor with the channel.

Cell membranes isolated from hamster insulinoma (HIT T15) cells at passages 65-74 contain high and low affinity receptors for a sulfonylurea derivative, 5-[125I]iodo,2-hydroxyglyburide (KD values of approximately 7 nM and 16 microM). Between passages 75 and 85, the estimated B(max) for the high affinity receptor decreases approximately 10-fold from approximately 1.6 to 0.16 pmol/mg membrane protein. By contrast, the density of low affinity binding sites, 800-1000 pmol/mg, is unaltered. The drop in high affinity receptors is paralleled by a decrease in the density of KATP channels assessed using patch-clamp and 86Rb(+)-efflux techniques. These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATP channel.