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W2022342405 abstract "Diversity of sulfonylurea receptor (SUR) subunits underlies tissue specific pharmacology of KATPchannels, which represent critical regulators of electrical activity in numerous cells. Notably, the neuronal/pancreatic β-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: Thr1059–Leu1087 and KCO II: Arg1218–Asn1320; 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 (Ile1088–Val1217) with the corresponding domain of SUR1. We infer the receptor sites for KCOs and SUs to be closely associated within a regulatory domain (Thr1059–Asn1320) in TMDII of SURs. Diversity of sulfonylurea receptor (SUR) subunits underlies tissue specific pharmacology of KATPchannels, which represent critical regulators of electrical activity in numerous cells. Notably, the neuronal/pancreatic β-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: Thr1059–Leu1087 and KCO II: Arg1218–Asn1320; 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 (Ile1088–Val1217) with the corresponding domain of SUR1. We infer the receptor sites for KCOs and SUs to be closely associated within a regulatory domain (Thr1059–Asn1320) in TMDII of SURs. Potassium channel openers (KCOs) 1The abbreviations used are:KCOpotassium channel openerKATPATP-sensitive K+ channelKCO I and KCO IIpotassium channel opener binding regionsKIRinwardly rectifying K+ channelNBFnucleotide binding foldSUsulfonylureaSUBRsulfonylurea binding regionSURsulfonylurea receptorTMDtransmembrane domainTMDI or TMDIIfirst (1Lawson K. Pharmacol. Ther. 1996; 70: 39-63Crossref PubMed Scopus (88) Google Scholar, 2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar, 3Aguilar-Bryan L. Clement J.P. IV Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (507) Google Scholar, 4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 7Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (242) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 9Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (416) Google Scholar, 11Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. ( Lond. ). 1997; 499: 715-720Crossref PubMed Scopus (334) Google Scholar) or second (12Chutkow W.A. Simon M.C. Le Beau M.M. Burant C.F. Diabetes. 1996; 45: 1439-1445Crossref PubMed Scopus (222) Google Scholar, 13Okuyama Y. Yamada M. Kondo C. Satoh E. Isomoto S. Shindo T. Horio Y. Kitakaze M. Hori M. Kurachi Y. Pflügers Arch. 1998; 435: 595-603Crossref PubMed Scopus (74) Google Scholar, 14Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Circ. Res. 1998; 83: 1132-1143Crossref PubMed Scopus (155) Google Scholar, 15Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar, 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 17Gribble F.M. Tucker S.J. Seino S. Ashcroft F.M. Diabetes. 1998; 47: 1412-1418Crossref PubMed Scopus (233) Google Scholar) set of transmembrane domains (see Fig. 1, A or F)DMEMDulbecco's modified Eagle's medium 1The abbreviations used are:KCOpotassium channel openerKATPATP-sensitive K+ channelKCO I and KCO IIpotassium channel opener binding regionsKIRinwardly rectifying K+ channelNBFnucleotide binding foldSUsulfonylureaSUBRsulfonylurea binding regionSURsulfonylurea receptorTMDtransmembrane domainTMDI or TMDIIfirst (1Lawson K. Pharmacol. Ther. 1996; 70: 39-63Crossref PubMed Scopus (88) Google Scholar, 2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar, 3Aguilar-Bryan L. Clement J.P. IV Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (507) Google Scholar, 4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 7Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (242) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 9Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (416) Google Scholar, 11Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. ( Lond. ). 1997; 499: 715-720Crossref PubMed Scopus (334) Google Scholar) or second (12Chutkow W.A. Simon M.C. Le Beau M.M. Burant C.F. Diabetes. 1996; 45: 1439-1445Crossref PubMed Scopus (222) Google Scholar, 13Okuyama Y. Yamada M. Kondo C. Satoh E. Isomoto S. Shindo T. Horio Y. Kitakaze M. Hori M. Kurachi Y. Pflügers Arch. 1998; 435: 595-603Crossref PubMed Scopus (74) Google Scholar, 14Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Circ. Res. 1998; 83: 1132-1143Crossref PubMed Scopus (155) Google Scholar, 15Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar, 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 17Gribble F.M. Tucker S.J. Seino S. Ashcroft F.M. Diabetes. 1998; 47: 1412-1418Crossref PubMed Scopus (233) Google Scholar) set of transmembrane domains (see Fig. 1, A or F)Figure 1Localization of the KCO receptor site on SURs. A, two regions within TMDII are essential for high affinity KCO binding. Schemata of chimeric constructs are shown on the left (for details see “Experimental Procedures”), and dissociation constants (KD values) for binding of P1075 are shown on the right of the figure. Displacement of [3H]P1075 (3 nm ( a )) or [3H]glibenclamide (0.3 nm ( b )) by unlabeled P1075 was assessed using membranes from COS-7 cells transiently expressing wild type isoforms or chimeras. KD values are shown as mean ± S.E. calculated from half-maximally inhibitory concentrations (IC50 values) of n = 4–16 independent displacement curves (see partB).c, p < 0.05 for the comparison with SUR2B. d, p < 0.05 for the comparison with SUR1. n.d., not detectable (affinity was too low for detection of specific [3H]P1075 or [3H]glibenclamide binding). KD values were: 11 ± 2 nm (SUR2B), 1.06 ± 0.1 mm (SUR1), 13 ± 2 nm (I), 14 ± 2 nm (II), 10 ± 1 nm (III), 31 ± 5 nm (IV), 48 ± 4 nm (V), 0.65 ± 0.08 mm (VIII), 0.24 ± 0.04 mm (IX), 0.17 ± 0.02 μm (X). B, binding affinities of KCOs for chimera X. [3H]P1075 (3 nm) displacement assays (n = 4–5) were done with membranes from COS-7 cells expressing chimera X. IC50 values and Hill coefficients are: 0.17 ± 0.02 μm, 0.91 (P1075, ●); 1.8 ± 0.2 μm, 0.98 (pinacidil, ▪); 10 ± 2 μm, 1.09 (levcromakalim, ▴). Curves for SUR2B taken from Ref. 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar. C, P1075-induced activation of channels reconstituted in COS-7 cells by expression of SUR subunits with KIR6.2 as indicated. Representative currents recorded from inside-out patches at −50 mV. Inward currents are shown as downward deflections. The patch was exposed to P1075 and ATP as indicated by the lines above the records. D, potencies of KCOs to open channels reconstituted with chimeras VII, X, or SUR1. Channel activation was recorded in inside-out patches as shown in partC. Results (n = 3–5) are expressed as percentage of maximal drug-induced channel activation (activity induced by 0.3 mm diazoxide, see Refs. 5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar and 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar). EC50values (half-maximally effective concentrations) and Hill coefficients are: 0.61 ± 0.12 μm, 1.41 (P1075, ●); 13 ± 5 μm, 1.28 (pinacidil, ▪); 52 ± 11 μm, 1.35 (levcromakalim, ▴). Curves for SUR2B taken from Ref. 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar. E, SU affinities of chimeras II and X. Displacement of [3H]glibenclamide (0.3 nm) by unlabeled compounds (Glib = glibenclamide; Glip = glipizide; Tolb = tolbutamide; n = 4–5) was done with membranes from COS-7 cells expressing chimeras as indicated. IC50 values and Hill coefficients are: 1.5 ± 0.2 nm, 1.02 (II, Glib, ●); 27 ± 4 nm, 0.94 (II, Glip, ▪); 24 ± 3 μm, 1.09 (II, Tolb, ▴); 0.98 ± 0.07 nm, 1.01 (X, Glib, ▿). Curves for SUR1 taken from Ref.18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar. F, putative transmembrane topologies of the regions essential for KCO (KCO I and II) and SU (SUBR) binding. Potential topology was assigned by hydropathy analysis (26Hopp T.P. Woods K.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3824-3828Crossref PubMed Scopus (2886) Google Scholar) assuming 17 TMDs (27Tusnady G.E. Bakos E. Varadi A. Sarkadi B. FEBS Lett. 1997; 402: 1-3Crossref PubMed Scopus (215) Google Scholar). Filled circles represent amino acids within KCO I and II (sequence numbers indicate first or last amino acid; rat SUR2B numbering). G, amino acid sequence alignment of KCO I and II in SUR2B and SUR1 (divergent amino acids shown). ic = intracellular; tm = transmembrane; ec = extracellular.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DMEMDulbecco's modified Eagle's medium comprise a structurally diverse group of drugs with a broad spectrum of potential therapeutic applications (e.g. hypoglycemia, hypertension, arrhythmias, angina pectoris, asthma) (1Lawson K. Pharmacol. Ther. 1996; 70: 39-63Crossref PubMed Scopus (88) Google Scholar). These drugs (e.g.P1075, pinacidil, levcromakalim, diazoxide) exert their effects on secretory cells, neurones, vascular and nonvascular smooth muscle, and on cardiac and skeletal muscle by opening ATP-sensitive potassium channels (KATP channels), thus shifting the membrane potential toward the reversal potential for potassium and reducing cellular electrical activity (2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar). potassium channel opener ATP-sensitive K+ channel potassium channel opener binding regions inwardly rectifying K+ channel nucleotide binding fold sulfonylurea sulfonylurea binding region sulfonylurea receptor transmembrane domain first (1Lawson K. Pharmacol. Ther. 1996; 70: 39-63Crossref PubMed Scopus (88) Google Scholar, 2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar, 3Aguilar-Bryan L. Clement J.P. IV Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (507) Google Scholar, 4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 7Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (242) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 9Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (416) Google Scholar, 11Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. ( Lond. ). 1997; 499: 715-720Crossref PubMed Scopus (334) Google Scholar) or second (12Chutkow W.A. Simon M.C. Le Beau M.M. Burant C.F. Diabetes. 1996; 45: 1439-1445Crossref PubMed Scopus (222) Google Scholar, 13Okuyama Y. Yamada M. Kondo C. Satoh E. Isomoto S. Shindo T. Horio Y. Kitakaze M. Hori M. Kurachi Y. Pflügers Arch. 1998; 435: 595-603Crossref PubMed Scopus (74) Google Scholar, 14Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Circ. Res. 1998; 83: 1132-1143Crossref PubMed Scopus (155) Google Scholar, 15Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar, 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 17Gribble F.M. Tucker S.J. Seino S. Ashcroft F.M. Diabetes. 1998; 47: 1412-1418Crossref PubMed Scopus (233) Google Scholar) set of transmembrane domains (see Fig. 1, A or F) Dulbecco's modified Eagle's medium potassium channel opener ATP-sensitive K+ channel potassium channel opener binding regions inwardly rectifying K+ channel nucleotide binding fold sulfonylurea sulfonylurea binding region sulfonylurea receptor transmembrane domain first (1Lawson K. Pharmacol. Ther. 1996; 70: 39-63Crossref PubMed Scopus (88) Google Scholar, 2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar, 3Aguilar-Bryan L. Clement J.P. IV Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (507) Google Scholar, 4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 7Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (242) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 9Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (416) Google Scholar, 11Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. ( Lond. ). 1997; 499: 715-720Crossref PubMed Scopus (334) Google Scholar) or second (12Chutkow W.A. Simon M.C. Le Beau M.M. Burant C.F. Diabetes. 1996; 45: 1439-1445Crossref PubMed Scopus (222) Google Scholar, 13Okuyama Y. Yamada M. Kondo C. Satoh E. Isomoto S. Shindo T. Horio Y. Kitakaze M. Hori M. Kurachi Y. Pflügers Arch. 1998; 435: 595-603Crossref PubMed Scopus (74) Google Scholar, 14Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Circ. Res. 1998; 83: 1132-1143Crossref PubMed Scopus (155) Google Scholar, 15Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar, 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 17Gribble F.M. Tucker S.J. Seino S. Ashcroft F.M. Diabetes. 1998; 47: 1412-1418Crossref PubMed Scopus (233) Google Scholar) set of transmembrane domains (see Fig. 1, A or F) Dulbecco's modified Eagle's medium Recent progress resulted in cloning of KATP channels and elucidation of their subunit composition (see Ref. 3Aguilar-Bryan L. Clement J.P. IV Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (507) Google Scholar for a review). These channels are assembled with a tetradimeric stoichiometry, (SUR/Kir6.x)4, from two structurally distinct subunits, an inwardly rectifying potassium channel subunit (KIR6.1 or KIR6.2) forming the pore and a regulatory subunit, a sulfonylurea receptor (SUR), belonging to the ATP-binding cassette superfamily with multiple transmembrane domains (TMDs) and two nucleotide binding folds (NBFs) (4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 7Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (242) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 9Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 10Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (416) Google Scholar, 11Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. ( Lond. ). 1997; 499: 715-720Crossref PubMed Scopus (334) Google Scholar). Three isoforms of SURs have been cloned, SUR1 and two splice products of a single gene, SUR2A and SUR2B, differing only in their C-terminal 42–45 amino acids (4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 12Chutkow W.A. Simon M.C. Le Beau M.M. Burant C.F. Diabetes. 1996; 45: 1439-1445Crossref PubMed Scopus (222) Google Scholar). SUR1/KIR6.2 have been proposed to reconstitute the neuronal/pancreatic β-cell (5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar), SUR2A/KIR6.2, the cardiac (6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 13Okuyama Y. Yamada M. Kondo C. Satoh E. Isomoto S. Shindo T. Horio Y. Kitakaze M. Hori M. Kurachi Y. Pflügers Arch. 1998; 435: 595-603Crossref PubMed Scopus (74) Google Scholar, 14Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. Circ. Res. 1998; 83: 1132-1143Crossref PubMed Scopus (155) Google Scholar), and SUR2B/KIR6.1 (or KIR6.2), the vascular smooth muscle-type KATP channels (8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 11Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. ( Lond. ). 1997; 499: 715-720Crossref PubMed Scopus (334) Google Scholar, 15Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar, 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar). Notably, diversity of SURs confers tissue-specific pharmacology, with SUR2 isoforms imparting high sensitivity to KCOs and low to sulfonylureas (SUs) and SUR1 mediating inverse sensitivities (5Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1597) Google Scholar, 6Inagaki N. Gonoi T. Clement J.P., IV Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 8Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar,16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 17Gribble F.M. Tucker S.J. Seino S. Ashcroft F.M. Diabetes. 1998; 47: 1412-1418Crossref PubMed Scopus (233) Google Scholar, 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar). Unraveling the molecular basis for these divergent drug sensitivities and understanding the mechanisms involved in drug-induced modulation of KATP channel activity is of key importance for design of tissue specific compounds. Here, we report two regions within the second set of transmembrane domains (TMDII) of SURs to be essential for KCO binding and action. [3H]P1075 (specific activity 116 Ci mmol−1) was purchased from Amersham Pharmacia Biotech Freiburg, Germany). [3H]Glibenclamide (specific activity 51 Ci mmol−1) was from NEN Life Science Products (Dreieich, Germany). All other chemicals and drugs were obtained from the sources described elsewhere (16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar, 19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Crossref PubMed Scopus (56) Google Scholar, 20Schwanstecher M. Schwanstecher C. Dickel C. Chudziak F. Moshiri A. Panten U. Br. J. Pharmacol. 1994; 113: 903-911Crossref PubMed Scopus (45) Google Scholar). Stock solutions of drugs were prepared in KOH (50 mm) or dimethyl sulfoxide with a final solvent concentration in the media below 1%. Chimeras comprising segments from hamster SUR1 (GenBankTM accession number A56248) or rat SUR2B (GenBankTM accession number AF087838) were constructed using standard molecular biology techniques. Products were subcloned into the pECE vector (4Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar) and sequenced to verify constructs and polymerase chain reaction fidelity before transfection. Composition of chimeras was as follows (numbers indicate amino acid boundaries of SUR2B or SUR1 as indicated; see also Fig. 1 A): chimera I (1–675, SUR2B)-(687–901, SUR1)-(880–1545, SUR2B); chimera II (1–1087, SUR2B)-(1121–1250, SUR1)-(1218–1545, SUR2B); chimera III (1–1320, SUR2B)- (1358–1582, SUR1); chimera IV (1–919, SUR2B)-(942–1091, SUR1)-(1059–1545, SUR2B); chimera V (1–686, SUR1)-(676–1545, SUR2B); chimera VI (1–1058, SUR2B)-(1092–1120, SUR1)-(1088–1545, SUR2B); chimera VII (1–1217, SUR2B)-(1251–1357, SUR1)-(1321–1545, SUR2B); chimera VIII (1–1091, SUR1)-(1059–1087, SUR2B)-(1121–1582, SUR1); chimera IX (1–1250, SUR1)-(1218–1320, SUR2B)-(1358–1582, SUR1); chimera X (1–1091, SUR1)-(1059–1087, SUR2B)-(1121–1250, SUR1)-(1218–1320, SUR2B)-(1358–1582, SUR1). Transfections and membrane preparations were performed as described previously (16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Crossref PubMed Scopus (56) Google Scholar). Briefly, COS-7 cells cultured in DMEM-HG (10 mm glucose), supplemented with 10% fetal calf serum, were plated at a density of 5 × 105cells per dish (94 mm) and allowed to attach overnight. 200 μg of pECE-SUR complementary DNA were used to transfect 10 plates. For transfection the cells were incubated 4 h in a Tris-buffered salt solution containing DNA (5–10 μg/ml) plus DEAE-dextran (1 mg/ml), 2 min in HEPES-buffered salt solution plus dimethyl sulfoxide (10%) and 4 h in DMEM-HG plus chloroquine (100 μm). Cells were then returned to DMEM-HG plus 10% fetal calf serum. Membranes were prepared 60–72 h posttransfection as described (19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Crossref PubMed Scopus (56) Google Scholar). For binding experiments resuspended membranes (final protein concentration 5–50 μg/ml) were incubated in Tris buffer (50 mm, pH 7.4) containing either [3H]P1075 (final concentration 3 nm, nonspecific binding defined by 100 μmpinacidil) or [3H]glibenclamide (final concentration 0.3 nm, nonspecific binding defined by 100 nmglibenclamide) and other additions as shown in the figure. The free Mg2+ concentration was kept close to 0.7 mm. In P1075 assays (Fig. 1, A and B), MgATP (0.1 mm) was added to incubation media to enable KCO binding (16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar). Low affinity P1075 binding to SUR1 isoforms (Fig. 1 A) was measured via allosteric displacement of [3H]glibenclamide as described previously (16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar). Incubations were carried out for 1 h at room temperature and were terminated by rapid filtration through Whatman GF/B filters. Transfections were performed as described above with the following modification. COS-7 cells were plated at a density of 8 × 104 cells per dish (35 mm). 20 μg of pECE-SUR complementary DNA and 20 μg of pECE-mouse KIR6.2 complementary DNA (GenBankTMD50581) were mixed and used to transfect six 35-mm plates. Experiments in the inside-out configuration of the patch-clamp technique were performed 1–2 days after transfection at room temperature as described previously (20Schwanstecher M. Schwanstecher C. Dickel C. Chudziak F. Moshiri A. Panten U. Br. J. Pharmacol. 1994; 113: 903-911Crossref PubMed Scopus (45) Google Scholar). Membrane patches were clamped at −50 mV. The intracellular bath solution contained (mm) 140 KCl, 2 CaCl2, 0.7 free Mg2+, 10 EGTA, 5 HEPES (pH 7.3) and the pipette solution 146 KCl, 2.6 CaCl2, 1.2 MgCl2, and 10 HEPES (pH 7.4). For registration of concentration-response curves (Fig.1 D) patches were chosen with little “run-down” over the measuring period and drug effects were corrected for this loss of channel activity by use of linear interpolation. Artifacts due to incomplete drug washout or slow reversibility were excluded by making sure that cumulative experiments with stepwise increase or decrease of the drug concentration yielded identical EC50 values and slope factors. Channel activity (A) was defined as the product of the number of functional channels (n) and the probability of the channels being in the open state (p). A was calculated by dividing the mean current (I) by the single-channel current amplitude (i). Density of KATP channels per patch ranged from 15 to 50. Varying channel densities did not affect EC50 values or Hill coefficients. Data analysis (including calculation ofKD values from IC50 values), and statistics were performed as described (19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Crossref PubMed Scopus (56) Google Scholar, 20Schwanstecher M. Schwanstecher C. Dickel C. Chudziak F. Moshiri A. Panten U. Br. J. Pharmacol. 1994; 113: 903-911Crossref PubMed Scopus (45) Google Scholar). Results shown as mean ± S.E. (n = 3–16). The pharmacological hallmark of SUR2B is its high affinity for KCOs, the KD for P1075 (rat; 11 ± 2 nm) being approximately 100,000-fold lower than that of SUR1 (hamster; 1.06 ± 0.1 mm; see also Ref. 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar). Based on this huge affinity difference the KCO receptor site was localized by systematically substituting corresponding domains between both isoforms (Fig. 1 A). Whereas both NBFs (chimera I and III; KD = 13 ± 2 nm or 10 ± 1 nm, respectively) and TMDs 14–15 (chimera II; KD = 14 ± 2 nm) did not contribute to discrepant affinities, small, 3–5-fold reductions of SUR2B's P1075 affinity were induced by replacing TMDs 12–13 (chimera IV; KD = 31 ± 5 nm) or 1–11 (chimera V; KD = 48 ± 4 nm), indicating these latter domains to interfere with the binding process either directly or indirectly. Two regions, part of the cytosolic loop between TMD 13 and 14 (KCO I: Thr1059–Leu1087; chimera VI) and TMDs 16–17 (KCO II: Arg1218–Asn1320; chimera VII), however, were identified to be essential, with complete loss of detectable [3H]P1075 binding resulting from substitution. Consistently, combined transfer of these domains into SUR1 induced a 6,200-fold increase of P1075 affinity (chimera X; KD= 0.17 ± 0.02 μm), whereas split substitutions mediated small, 1.5–4-fold enhancements (chimera VIII and IX;KD = 0.65 ± 0.08 mm or 0.24 ± 0.04 mm, respectively), implying both domains to interact in formation of the KCO binding site. Strong gain of P1075 affinity in chimera X was paralleled by affinity increments for pinacidil (270 fold; KD = 1.8 ± 0.2 μm) and levcromakalim (>50-fold;KD = 10 ± 2 μm), thus reconstituting the SUR2B characteristic rank order (Fig. 1 B; see Ref. 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar for KCO binding to SUR1). Binding was matched by drug action (Fig. 1, C and D), with P1075 sensitivity of channels reconstituted with the loss-of-affinity constructs (chimera VI or VII) resembling that mediated by SUR1 (EC50 > 1 mm; results shown for chimera VII only) and the gain-of-affinity chimera X conferring potencies (P1075, EC50 = 0.61 ± 0.12 μm; pinacidil, EC50 = 13 ± 5 μm; levcromakalim, EC50 = 52 ± 11 μm) similar to wild type SUR2B. Notably, glibenclamide affinity of chimera X (KD = 0.68 ± 0.05 nm; Fig. 1 E) equaled that of SUR1 (KD = 0.72 nm; Ref. 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar), suggesting KCO I and II not to form part of the SU receptor site and indicating that high affinity binding of SUs and KCOs can be combined within the same isoform. A region overlapping that separating KCO I and II was recently reported to be critical for SU sensitivity (21Ashfield R. Gribble F.M. Ashcroft S.J.H. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar). Consistently, we found substitution of this linking region (SUBR: Ile1088-Val1217; Fig. 1 F) by the corresponding domain of SUR1 (Ile1121–Val1250; chimera II) to significantly enhance SU affinities (210-fold for glibenclamide, KD = 1.2 ± 0.15 nm; 280-fold for glipizide, KD = 22 ± 3 nm; 14-fold for tolbutamide, KD = 19 ± 2 μm; see Ref. 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar for SU binding to SUR 2B) with dissociation constants resembling that of wild type SUR1 (Fig.1 E). Similar to KCOs, affinity increments were paralleled by drug action. Glibenclamide sensitivity of channels transiently reconstituted from chimera II plus KIR6.2 (EC50= 0.22 ± 0.09 nm; n = 4; results not shown in a figure) was 190-fold higher than that of SUR2B/KIR6.2 channels (EC50 = 42 nm; Ref. 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar) coinciding with the drug's potency to inhibit activity of SUR1/KIR6.2 channels (EC50 = 0.13 nm; Ref. 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar). Expression rates of the chimeras did not differ markedly from that of the wild type receptors, ranging from 10 to 50 pmol/mg of membrane protein as calculated from maximal number of binding sites (chimeras I–V and VIII–X) or estimated from reconstituted channel activity (chimeras VI and VII). This study is the first to localize regions in SURs critical for formation of the KCO binding pocket and to establish that high affinity KCO and SU binding can coexist within the same isoform. These conclusions are based on the following findings. 1) Substitution of two regions within TMDII of SUR2B (KCO I and II, Fig. 1 F) with the corresponding domains of SUR1 (chimera VI and VII) induced a complete loss of detectable [3H]P1075 binding (Fig.1 A). 2) Simultaneous transfer of these regions into SUR1 (chimera X) strongly increased KCO affinities (6,200-fold for P1075) reproducing the SUR2B characteristic binding pattern for P1075, pinacidil, and levcromakalim (Fig. 1, A and B). 3) High glibenclamide affinity of SUR1 was not reduced by this transfer (Fig. 1 E). 4) Loss or gain of KCO affinity were paralleled by corresponding sensitivity changes of channels reconstituted with KIR6.2 (Fig. 1, C and D). The regions critical for KCO binding reside in TMDII forming part of the putative intracellular loop connecting TMD 13 and 14 (KCO I: Thr1059–Leu1087, SUR2 numbering) and the domain preceding NBF2 (KCO II: Arg1218–Asn1320, SUR2 numbering) (Fig.1 F). Either of the two regions proved essential for reconstitution of the SUR2B characteristic pattern of high KCO affinities, strongly arguing that both domains interact in formation of the binding pocket. However, since TMDs 1–11 (chimera V) and 12–13 (chimera IV) were required for full KCO affinity, additional regions might be involved (Fig. 1 A). Similarly, we have shown recently that the C-terminal 42 amino acids affect KCO affinity by a factor of 3–5 with SUR1 = SUR2B > SUR2A (16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar). Identification of KCO I in a putative intracellular loop suggests localization of the receptor site at the internal face of the plasma membrane implying that, equivalent to SUs (20Schwanstecher M. Schwanstecher C. Dickel C. Chudziak F. Moshiri A. Panten U. Br. J. Pharmacol. 1994; 113: 903-911Crossref PubMed Scopus (45) Google Scholar), KCOs have to cross the membrane to exert their effect. This finding also hints the putative intracellular part of KCO II (Ala1266–Asn1320, SUR2B numbering; Fig. 1, F and G) to act as counterpart of KCO I in formation of the site. Albeit, limiting substitutions to this part of KCO II (plus KCO I) did not lead to reconstitution of high KCO affinity (results not shown). Importantly, high SU affinity could be conferred on SUR2B by substituting the region separating KCO I and II (SUBR: Ile1088–Val1217, SUR2 numbering; Fig.1 F) by the corresponding domain of SUR1 (chimera II). Since this transfer did perfectly reconstitute affinities of SUR1 for glibenclamide, glipizide, and tolbutamide (Fig. 1 E), the results strongly suggest SUBR to form the SU binding site, thus supporting conclusions from a recent study (21Ashfield R. Gribble F.M. Ashcroft S.J.H. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar). We infer TMDs 14–17 (Thr1059–Asn1320, SUR2 numbering) within TMDII of SURs to be of key importance for drug-induced KATP channel modulation with the core region of this regulatory domain forming the binding site for SUs (SUBR) and the flanking regions (KCO I and KCO II) constituting main parts of the receptor site for KCOs (Fig. 1 F). The idea of distinct (although closely associated) sites is supported by substitution of SUBR lacking an effect on P1075 affinity (chimera II, Fig.1 A) and transfer of KCO I and II not affecting theKD for glibenclamide (chimera X, Fig. 1,A and E). Close local association of SU and KCO binding regions, on the other hand, conforms with evidence for negative allosteric coupling of the sites (15Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar, 16Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (195) Google Scholar, 18Dörschner H. Brekardin E. Uhde I. Schwanstecher C. Schwanstecher M. Mol. Pharmacol. 1999; 55: 1060-1066Crossref PubMed Scopus (89) Google Scholar, 19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Crossref PubMed Scopus (56) Google Scholar, 22Schwanstecher M. Löser S. Rietze I. Panten U. Naunyn Schmiedeberg's Arch. Pharmacol. 1991; 343: 83-89Crossref PubMed Scopus (55) Google Scholar, 23Quast U. Bray K.M. Andres H. Manley P.W. Baumlin Y. Dosogne J. Mol. Pharmacol. 1993; 43: 474-481PubMed Google Scholar, 24Löffler C. Quast U. Br. J. Pharmacol. 1997; 120: 476-480Crossref PubMed Scopus (28) Google Scholar). Notably, pharmacological properties of SUR1 and SUR2B were combined in either chimera II and X (Fig. 1, A and E), thus establishing for the first time high affinity for KCOs and SUs not to be mutually exclusive. Hence, native SUR isoforms with similar properties might exist, and accordingly evidence for a receptor with high P1075 and glibenclamide affinity has been presented recently in vascular smooth muscle (25Löffler-Walz C. Quast U. Naunyn Schmiedeberg's Arch. Pharmacol. 1998; 357: 183-185Crossref PubMed Scopus (14) Google Scholar). Both chimeras provide excellent tools for further analysis of functional interaction between the drug sites. High KCO and SU affinities of chimeras II and X matched sensitivities of channels transiently reconstituted with KIR6.2 (see “Results” and Fig. 1, B–E). This finding implies that SUR isoforms use identical mechanisms to transduce drug binding to the regulatory domain (KCO I + SUBR + KCO II; Thr1059–Asn1320, SUR2 numbering; Fig.1 F) into modulation of channel activity. It might be argued that the regulatory domain does not form the receptor sites itself but indirectly affects KCO or SU binding in other regions. Although this possibility cannot be ruled out, it is unlikely to explain restitution of the correct rank order of affinities (Fig. 1,B and E). The study provides new insight into the molecular mechanisms of drug-induced KATP channel regulation. We conclude the receptor sites for KCOs and SUs to be closely associated within a regulatory domain in TMDII of SURs. We are grateful to Dr. Lydia Aguilar-Bryan and Dr. Joseph Bryan (Baylor College of Medicine, Houston, TX) for the hamster SUR1 clone and stimulating discussions. We thank Haide Fürstenberg, Ursula Herbort-Brand, Gisela Müller, Claudia Ott, Beate Pieper, and Carolin Rattunde for excellent technical assistance." @default.
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W2022342405 title "Identification of the Potassium Channel Opener Site on Sulfonylurea Receptors" @default.
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