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The Stereoenantiomers of a Pinacidil Analog Open or Close Cloned ATP-sensitive K+ Channels

2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês

10.1074/jbc.m206685200

1083-351X

U Lange, Cornelia Löffler‐Walz, Heinrich Englert, Annette Hambrock, Ulrich Ruß, Ulrich Quast,

Anesthesia and Neurotoxicity Research

ATP-dependent K+channels (KATP channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening KATP channels. In the vasculature, openers of KATP channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be KATP channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, theR-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC50 ∼50 nm in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that theR-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas theS-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a KATP channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR. ATP-dependent K+channels (KATP channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening KATP channels. In the vasculature, openers of KATP channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be KATP channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, theR-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC50 ∼50 nm in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that theR-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas theS-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a KATP channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR. ATP-dependent K+ channels (KATP channels) 1The abbreviations used are: KATP channel, ATP-sensitive K+ channel; P1075, N-cyano-N′-(1,1-dimethylpropyl)-N“-3-pyridylguanidine; Kir, inwardly rectifying K+ channel; SUR, sulfonylurea receptor; HEK, human embryonic kidney; PNU-96293, (R)-N-cyano-N′-(1-phenylpropyl)-N”-3-pyridylguanidine; PNU-96179, (S)-N-cyano-N′-(1-phenylpropyl)- N“-3-pyridylguanidine. are gated by the intracellular ATP/ADP ratio with ATP inducing channel closure and MgADP channel opening. Functionally, these channels link membrane potential and excitability to the metabolic state of the cell (1Ashcroft S.J.H. Ashcroft F.M. Cell. Signal. 1990; 2: 197-214Google Scholar, 2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Google Scholar). Pharmacologically, KATP channels are closed by the hypoglycemic sulfonylureas such as glibenclamide and their benzoic acid analogs, the glinides; these drugs are used in the treatment of diabetes type 2. KATP channels are activated by the K+ channel openers, a chemically heterogeneous group of compounds including the cyanoguanidines like pinacidil and P1075 and the benzopyrans such as levcromakalim; these compounds relax smooth muscle and induce hypotension (2Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Google Scholar, 3Lawson K. Kidney Int. 2000; 57: 838-845Google Scholar, 4Coghlan M.J. Carroll W.A. Gopalakrishnan M. J. Med. Chem. 2001; 44: 1627-1653Google Scholar, 5Gribble F.M. Reimann F. Biochem. Soc. Trans. 2002; 30: 333-339Google Scholar). KATP channels are heteromeric complexes composed of pore-forming inwardly rectifying K+ channels (Kir6.x) and sulfonylurea receptors (SURs) (6Inagaki N. Gonoi T. Clement IV J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Google Scholar, 7Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Google Scholar) (for reviews, see Refs. 8Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Google Scholar, 9Aguilar-Bryan L. Bryan J. Endocrine Rev. 1999; 20: 101-135Google Scholar, 10Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Google Scholar). Kir6.x is encoded by either one of two genes giving rise to two subtypes, Kir6.1 and -6.2, with the latter being present in most KATP channels. ATP binding to Kir6.2 induces channel closure, thus accounting for the inhibitory action of the nucleotide (11Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Google Scholar). SUR is a member of the ATP-binding cassette protein superfamily (7Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Google Scholar, 12Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzáles G. Herrera-Soza H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Google Scholar). It carries binding sites for nucleotides, the nucleotide binding folds (13Ueda K. Komine J. Matsuo M. Seino S. Amachi T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1268-1272Google Scholar), and for the sulfonylureas and the openers (12Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. Gonzáles G. Herrera-Soza H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Google Scholar, 14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar,15Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Google Scholar). SUR also is encoded by either one of two genes. SUR1, expressed in the pancreatic β-cell and predominantly in neurons, exhibits high affinity for the sulfonylureas and low affinity for the openers. SUR2 is expressed in the various muscle types and shows high affinity for the openers but lower affinity for the sulfonylureas (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar,15Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Google Scholar) (for reviews, see Refs. 5Gribble F.M. Reimann F. Biochem. Soc. Trans. 2002; 30: 333-339Google Scholar and 16Ashcroft F.M. Gribble F.M. Trends Pharmacol. Sci. 2000; 21: 439-445Google Scholar). Alternative splicing of the SUR2 mRNA leads to two isoforms, SUR2A and SUR2B, which differ only in the last carboxyl-terminal exon. SUR2A is expressed in skeletal and heart muscle; SUR2B is expressed in smooth muscle (17Inagaki N. Gonoi T. Clement IV J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Google Scholar, 18Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Google Scholar). The nucleotides not only gate the channel, they also profoundly affect the potency and efficacy of the synthetic KATP channel modulators by allosteric interactions. First, there is a positive allosteric interaction between nucleotide and opener binding, enabling SUR to bind openers with high affinity (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar, 15Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Google Scholar, 16Ashcroft F.M. Gribble F.M. Trends Pharmacol. Sci. 2000; 21: 439-445Google Scholar, 19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Google Scholar, 20Quast U. Bray K.M. Andres H. Manley P.W. Baumlin Y. Dosogne J. Mol. Pharmacol. 1993; 43: 474-481Google Scholar). Conversely, openers enhance the ATPase activity of SUR2A, thereby promoting channel activation (21Bienengraeber M. Alekseev A.E. Abraham M.R. Carrasco A.J. Moreau C. Vivaudou M. Dzeja P.P. Terzic A. FASEB J. 2000; 14: 1943-1952Google Scholar). Second, there is a negative allosteric interaction between nucleotide and sulfonylurea binding (22Schwanstecher M. Löser S. Rietze I. Panten U. Naunyn-Schmiedeberg's Arch. Pharmacol. 1991; 343: 83-89Google Scholar, 23Hambrock A. Löffler-Walz C. Quast U. Br. J. Pharmacol. 2002; 136: 995-1004Google Scholar). Third, there is a negative allosteric coupling between opener and sulfonylurea binding, generally leading to mutually exclusive binding of the two ligands to SUR (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar, 15Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Google Scholar, 19Schwanstecher M. Brandt C. Behrends S. Schaupp U. Panten U. Br. J. Pharmacol. 1992; 106: 295-301Google Scholar, 24Bray K.M. Quast U. J. Biol. Chem. 1992; 267: 11689-11692Google Scholar). In varying the cyanoguanidine structure, Khan and colleagues (25Khan S.A. Higdon N.R. Hester J.B. Meisheri K.D. J. Pharmacol. Exp. Ther. 1997; 283: 1207-1213Google Scholar) discovered that introduction of a phenyl ring in the side chain (Fig.1) gave compounds that potently reversed the vasodilation produced by various KATP channel openers but not that by other vasodilatory maneuvers; in addition, the inhibitory effect resided in one (i.e. the R-) enantiomer. It was concluded that the novel compounds acted as KATP channel blockers. The results of their study may be interpreted in two ways. First, the compounds may have acted as “neutral antagonists” (i.e.by displacing the opener from SUR without directly affecting channel activity); channel closure would then be induced by the high intracellular ATP concentrations in the vascular smooth muscle cell. Alternatively, the compounds may have acted as “inverse agonists” (i.e. they displaced the opener, and, similar to the sulfonylureas, their binding induced channel block also in the absence of ATP). In order to decide between these alternatives, the enantiomers of one such compound, PNU-94750 (Fig. 1), were examined in electrophysiological experiments using recombinant KATP channels. Since KATP channel openers and blockers (such as sulfonylureas) differ in their allosteric coupling to MgATP binding (see above), we also investigated the effect of MgATP on the binding of the enantiomers in radioligand binding assays. The study showed that the enantiomers of PNU-94750 differed in their coupling to MgATP binding and that the R-enantiomer (PNU-96293) was negatively coupled and inhibited KATP channels, whereas theS-enantiomer (PNU-96179) was positively coupled and was an opener. SUR2B(Y1206S) was constructed from murine SUR2B (GenBankTMD86038 (18Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Google Scholar)) as described (26Hambrock A. Löffler-Walz C. Russ U. Lange U. Quast U. Mol. Pharmacol. 2001; 60: 190-199Google Scholar). Human embryonic kidney (HEK) 293 cells were cultured in minimum essential medium containing glutamine and supplemented with 10% fetal bovine serum and 20 μg/ml gentamycin (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar). Cells were transfected with the expression vector pcDNA3.1 (Invitrogen) containing the coding sequence of murine SUR2B, SUR2B(Y1206S), murine SUR2A (GenBankTMD86037 (18Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Google Scholar)), or rat SUR1 (GenBankTMX97279), and cell lines stably expressing these proteins were generated as described (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar). Cotransfection of SUR with murine Kir6.2 (D50581(17Inagaki N. Gonoi T. Clement IV J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Google Scholar)) was done transiently at a molar plasmid ratio of 1:1 using LipofectAMINE and Opti-MEM (Invitrogen) as described previously (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar). In cotransfections prepared for electrophysiological experiments, the pEGFP-C1 vector (CLONTECH, Palo Alto, CA), encoding for green fluorescent protein, was added for easy identification of transfected cells. 2–4 days after transfection, cells were used for binding studies and electrophysiological experiments. The patch-clamp technique was used in the inside-out, the cell-attached, and the whole-cell configuration as described by Hamill et al. (27Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. Eur. J. Physiol. 1981; 391: 85-100Google Scholar). Patch pipettes were drawn from borosilicate glass capillaries (GC 150 or GC 150T; Harvard Apparatus, Edenbridge, UK) and heat-polished using a horizontal microelectrode puller (Zeitz, Augsburg, Germany). For experiments using inside-out patches, bath and pipette were filled with a high K+-Ringer solution containing 142 mmKCl, 2.8 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 11 mmd-(+)-glucose; 10 mm HEPES, titrated to pH 7.4 with NaOH at 22 °C. After filling with buffer, pipettes had a resistance of 1–1.5 megaohms. After excision of the patch, the pipette was moved in front of a pipe with a high K+-EGTA-Ringer solution containing 143 mm KCl, 0.85 mmMgCl2, 1 mm CaCl2, 5 mmEGTA, 11 mmd-(+)-glucose, 10 mmHEPES, titrated to pH 7.2 with NaOH at 22 °C. The PNU compounds and glibenclamide were dissolved in stock solutions as described under “Materials”; ATP was dissolved in buffer, and an appropriate amount of MgCl2 was added to keep free Mg2+ constant at 0.7 mm. Substances were applied to the patch via the pipe, and patches were clamped to −50 mV. For evaluation of the inhibition by PNU-96293, traces were individually corrected for run down. For experiments in the cell-attached configuration, bath and pipette were filled with the high K+-Ringer solution described above. After filling, the pipettes had a resistance of 1–1.5 megaohms. Patch experiments were performed at 37 °C, and patches were clamped at −50 mV. Experiments using the whole-cell configuration were performed as described by Russ et al. (28Russ U. Hambrock A. Artunc F. Löffler-Walz C. Horio Y. Kurachi Y. Quast U. Mol. Pharmacol. 1999; 56: 955-961Google Scholar). The bath solution was 142 mm NaCl, 2.8 mm KCl, 1 mmMgCl2, 1 mm CaCl2, 11 mmd-(+)-glucose, 10 mm HEPES, titrated to pH 7.4 with NaOH at 37 °C. Patch pipettes were filled with 132 mm potassium glutamate, 10 mm NaCl, 2 mm MgCl2, 10 mm HEPES, 1 mm EGTA, 1 mm Na2ATP, titrated to pH 7.2 with NaOH, and had a resistance of 3–5 megaohms. Isolated cells were chosen. After establishing the whole cell configuration, cells were clamped to −60 mV, and every 12.5 s, the following square pulse protocol was applied. After 1 s at −60 mV, cells were clamped to the test potential of −110 mV for 0.5 s and then, after 1 s at −60 mV, to the next test potential of −90 mV, etc. (seven steps increasing by 20 mV from −110 to 10 mV). At the end of the protocol, capacitance and series resistance were measured, and the latter was compensated by 70%. To show the time-dependent current at a specified voltage, data were averaged over the last 125 ms spent at that voltage during the cycle and spliced together to give a continuous curve. Data were recorded with an EPC 9 amplifier (HEKA, Lambrecht, Germany) using the “Pulse” software (HEKA). Signals were filtered at 200 Hz using the four-pole Bessel filter of the EPC9 amplifier and sampled with 1 kHz. For cells stably expressing SUR alone, the antibiotic was withdrawn from the culture medium 1 week prior to membrane preparation. Membranes were prepared as described (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar). Protein concentration was determined according to Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Google Scholar) using bovine serum albumin as the standard. In equilibrium binding assays, membranes (final protein concentration 0.2–0.5 mg/ml) were added to the incubation buffer containing 139 mm NaCl, 5 mm KCl, 5 mm HEPES, 2.2/1.2 mm MgCl2, 1/0.003 mmNa2ATP and supplemented with the radioligand ([3H]glibenclamide ∼2.5 nm or [3H]P1075 ∼2 nm) and the inhibitor of interest at 37 °C. In the Mg2+-free experiments, Mg2+ was omitted from the incubation solution, and EDTA (1 mm) was added. At equilibrium (15 min for [3H]glibenclamide and 30 min for [3H]P1075 binding), incubation was stopped by diluting 0.3-ml aliquots in triplicate into 8 ml of ice-cold quench solution (50 mmTris-(hydroxymethyl)-aminomethane, 154 mm NaCl, pH 7.4) and rapid filtration under vacuum over Whatman GF/B filters (Whatman, Clifton, NJ). Filters were washed twice with 8 ml of ice-cold quench solution and counted for 3H in the presence of 6 ml of scintillant (Ultima Gold; Packard Instrument Co., Meriden, CT). Nonspecific binding of [3H]P1075 was determined in the presence of 10 μm P1075, and that of [3H]glibenclamide was determined in the presence of 100 μm P1075 or 1 μm glibenclamide for SUR2B(Y1206S) and SUR1, respectively. For measurement of the dissociation kinetics, membranes were incubated with 2 nm [3H]P1075 in the presence of 2.2 mm MgCl2 and 1 mmNa2ATP at 37 °C for 30 min. Dissociation was initiated by the addition of P1075 + PNU-96293 (10 μm each), and 300-μl aliquots were withdrawn at different times for filtration as described above. Experiments were conducted at 37 °C with an incubation time of 30 min as described previously (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar, 28Russ U. Hambrock A. Artunc F. Löffler-Walz C. Horio Y. Kurachi Y. Quast U. Mol. Pharmacol. 1999; 56: 955-961Google Scholar). Cells were suspended by rinsing with a HEPES-buffered physiological salt solution containing 139 mm NaCl, 5 mm KCl, 1.2 mmMgCl2, 1.25 mm CaCl2, 11 mmd-(+)-glucose, 5 mm HEPES; gassed with 95% O2 and 5% CO2; and titrated to pH 7.4 with NaOH at 37 °C. Binding experiments were started by the addition of cells (final concentration 1 × 106cells/ml, corresponding to 0.25 mg of protein/ml) to the buffer supplemented with ∼2 nm [3H]P1075 and the inhibitor of interest. After 30 min, incubation was stopped as described above, and aliquots were filtered over Whatman GF/C filters. Concentration dependencies were analyzed by fitting the logistic function y=100−A/(1+10n(px−pIC50))Equation 1 to the data. Here, A denotes the extent of the effect (amplitude); n (= nH) is the Hill coefficient, x is the concentration of the compound under study, and IC50 the midpoint of the curve withpx = −log x andpIC50 = −log IC50. In binding experiments, the dependence of the midpoint of an inhibition curve (IC50 value) on the concentration of the radioligand,L*, was calculated according to the equation by Cheng and Prusoff (30Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Google Scholar), IC50=Ki(1+L*/KL)Equation 2 where K i is the inhibition constant andK L is the equilibrium dissociation constant of the radioligand, L*. In general, this correction did not exceed a factor of 1.5. In cases when a homologous competition experiment (e.g. L* − L) was conducted in the presence of a fixed concentration of a further ligand,C, that also competed with the radioligand, Equation 2 was expanded to read as follows, IC50=KL(1+C/KC)+L*Equation 3 where K C is the equilibrium dissociation constant of competitor C. The homologous competition curves (in the absence and presence of competitor) may be transformed according to Scatchard (31Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Google Scholar), B/F=IC50/(Bmax−B)Equation 4 where B and F represent the bound and free concentration of the unlabeled ligand, L. B (in percent) is calculated from the radioactive ligand specifically bound,B*, as B = 100 − B*. Fits of the equations to the data were performed according to the method of least squares using the program SigmaPlot 6.1 (SPSS Science, Chicago, IL). Individual binding competition experiments were analyzed according to Equation 1. Errors in the parameters derived from the fit to a single curve were estimated using the univariate approximation (32Draper N.B. Smith H. Applied Regression Analysis. John Wiley & Sons, Inc., New York1981: 458-517Google Scholar). Amplitudes and pK values are normally distributed (33Christopoulos A. Trends Pharmacol. Sci. 1998; 19: 351-357Google Scholar); here, K values with the 95% confidence interval in parentheses are given. Propagation of errors was taken into account according to Bevington (34Bevington P.R. Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill, New York1969: 56-64Google Scholar). PNU-96293 and PNU-96179 were synthesized according to Humphrey et al. (35Humphrey, S. J., Meisheri, K. D., Ludens, J. H., and Hester, J. B. (October 22, 1996) U.S. Patent US5567722.Google Scholar). [3H]P1075 (specific activity 4.5 TBq (118 Ci) mmol−1) was purchased from Amersham Biosciences, and [3H]glibenclamide (specific activity 1.85 TBq (50 Ci) mmol−1) was from PerkinElmer Life Sciences. The reagents and media used for cell culture and transfection were from Invitrogen (Karlsruhe, Germany). Na2ATP was from Roche Diagnostics, and glibenclamide was from Sigma. P1075 was a kind gift from Leo Pharmaceuticals (Ballerup, Denmark). KATP channel modulators were dissolved in dimethyl sulfoxide/ethanol (1:1) and further diluted with the same solvent or with incubation buffer. In binding studies, the final solvent concentration in the assays was always below 0.3% (in electrophysiological experiments 0.1% or below). Khan and colleagues (25Khan S.A. Higdon N.R. Hester J.B. Meisheri K.D. J. Pharmacol. Exp. Ther. 1997; 283: 1207-1213Google Scholar) have shown that PNU-96293, theR-enantiomer, reversed the vasorelaxation induced by other K+ channel openers. We therefore examined the effect of this compound on the recombinant KATP channel in (nonvascular) smooth muscle, Kir6.2/SUR2B. After application of PNU-96293 (10, 30, and 100 μm) to an inside-out patch in the presence of 1 mm MgATP, no current developed (data not shown). In agreement with Khan et al. (25Khan S.A. Higdon N.R. Hester J.B. Meisheri K.D. J. Pharmacol. Exp. Ther. 1997; 283: 1207-1213Google Scholar), this indicated that the compound was devoid of opener activity. When the channel was opened by reducing the ATP concentration to 3 μm, the compound inhibited the channel concentration-dependently (Fig. 2 A). At 100 μm, the highest concentration tested, inhibition was ∼50%. In comparison, glibenclamide, at the saturating concentration of 1 μm, inhibited the current by 80%. The inhibition curve of the PNU enantiomer extrapolated to a maximum inhibition of 46% with an IC50 value of 16 μm (Fig.2 B, Table I). In the absence of ATP, the current was inhibited maximally by 30% with an IC50 value of 12 μm (Table I).Table IInhibition of recombinant KATP channels by PNU-96293ChannelConfigurationMgATPIC50ABlock by GBC (1 μm)mmμm%%Kir6.2/SUR2Bi/o012 (2, 83)30 ± 778 ± 5 (12)i/o0.00316 (7, 32)46 ± 677 ± 3 (15)Whole cell10.078 (0.037, 0.165)74 ± 696 ± 4 (5)Kir6.2/SUR2B(Y1206S)Whole cell10.045 (0.015, 0.140)86 ± 9100 ± 0 (3)aRef. 26.Kir6.2/SUR2Ai/o0.00321 (12, 37)54 ± 484 ± 5 (6)Whole cell10.014 (0.008, 0.023)bParameters for Kir6.2/SUR2A in the whole cell configuration refer to the group with high glibenclamide sensitivity (see “Results”).68 ± 4bParameters for Kir6.2/SUR2A in the whole cell configuration refer to the group with high glibenclamide sensitivity (see “Results”).79 ± 6 (3)bParameters for Kir6.2/SUR2A in the whole cell configuration refer to the group with high glibenclamide sensitivity (see “Results”).Kir6.2/SUR1i/o0At 100 μm40 ± 542 ± 6 (5)cInhibition by 100 μM tolbutamide.Experiments were performed as shown in Figs. Figure 2, Figure 3, Figure 4. The logistic function (Equation 1) with Hill coefficient 1 was fitted to the data. IC50 values are followed by the 95% confidence interval in parentheses. A, the extent of inhibition (amplitude); i/o, the inside-out configuration.a Ref. 26Hambrock A. Löffler-Walz C. Russ U. Lange U. Quast U. Mol. Pharmacol. 2001; 60: 190-199Google Scholar.b Parameters for Kir6.2/SUR2A in the whole cell configuration refer to the group with high glibenclamide sensitivity (see “Results”).c Inhibition by 100 μM tolbutamide. Open table in a new tab Experiments were performed as shown in Figs. Figure 2, Figure 3, Figure 4. The logistic function (Equation 1) with Hill coefficient 1 was fitted to the data. IC50 values are followed by the 95% confidence interval in parentheses. A, the extent of inhibition (amplitude); i/o, the inside-out configuration. The effect of PNU-96293 was also studied in the whole-cell configuration at 37 °C (Fig. 3). Dialysis of the cell with 1 mm MgATP generated a current that was inhibited by the PNU enantiomer in the nanomolar concentration range (IC50 = 78 nm) with a maximum inhibition of 74%; glibenclamide (1 μm) induced almost total inhibition (Table I). Analogous experiments using the Kir6.2/SUR2B(Y1206S) channel gave very similar results (Table I), showing that the mutation did not alter the sensitivity of the channel to PNU-96293. Fig. 4 Apresents an original recording from an inside-out patch containing Kir6.2/SUR2B channels. The channel was kept closed by MgATP (1 mm); the addition of PNU-96179 (100 μm) generated a current that was completely inhibited by glibenclamide (3 μm) in a reversible manner. Further experiments showed that the current induced by PNU-96179 (100 μm) was about 10–20% of that produced by P1075 at the maximally effective concentration of 0.1 μm; lower concentrations of the PNU compound showed smaller and inconsistent effects. The ability of PNU-96179 to open the Kir6.2/SUR2B channel was also examined in the whole-cell configuration; however, it proved difficult to keep the channel closed during prolonged dialysis of the cell with 3–10 mm MgATP. Experiments were therefore performed in the cell-attached configuration. Fig. 4 B shows that PNU-96179 (100 μm) activated the channels to 13% (mean 15.6 ± 1.6; n = 6) of the level produced by P1075 (0.1 μm); at 10 μm, the PNU compound was ineffective. Collectively, the data show that PNU-96179 is an opener of the Kir6.2/SUR2B channel, albeit with low potency and efficacy. The opening properties were not investigated further, since channel opening is an effect expected for a cyanoguanidine. Fig. 5illustrates the inhibition of [3H]P1075 binding to SUR2B by the two enantiomers in membranes. In the presence of 1 mm MgATP, the compounds inhibited binding of the radioligand up to 100% with Hill coefficients of 1 andK i values of 2.3 and 0.66 μm for PNU-96179 and PNU-96293, respectively (TableII). Hence, stereoselectivity of binding, measured as the eudismic (= K i) ratio, was weak (3.5). Similar experiments were performed also at an ATP concentration of 3 μm (i.e. the lowest concentration at which high affinity binding of the radioligand (which requires MgATP) is detected) (14Hambrock A. Löffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Google Scholar, 15Schwanstecher M. Sieverding C. Dörschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Google Scholar). Under these conditions, the inhibition curve for PNU-96179 was shifted to the right by a factor of 2.7 and that of PNU-96293 to the left by a factor of 10, giving now a eudismic ratio of 100. Hence, increasing ATP increased the potency of PNU-96179 and strongly decreased that of PNU-96293, indicating positive allosteric coupling between PNU-96179 and MgATP binding, whereas for PNU-96293, the coupling to MgATP was negative.Table IIBinding of PNU enantiomers to SUR subtypes and coupling to MgATP bindingPreparationRadioligandMgATPPNU-96179PNU-96293K iAaPercentage of binding specificity.K iAaPercentage of binding specificity.mMμm%μm%SUR2B[3H]P10751.02.3 (2.0, 2.7)1000.66 (0.58, 0.75)1000.0036.3 (4.0, 10)1000.065 (0.054, 0.078)100SUR2B/Kir6.2 (cells)[3H]P10753–5bAssumed intracellular MgATP concentration.3.6 (2.9, 4.6)1000.20 (0.16, 0.25)100SUR2B(Y1206S)[3H]GBC1.02.5 (1.0, 6.2)39 ± 21.7 (0.7, 4.3)−41 ± 3cThe negative sign indicates an increase in binding (to 141 and 121% of control, respectively (cf. Equation 1)).0.083 (58, 120)48 ± 60.018 (0.015, 0.020)38 ± 1SUR2A[3H]P10751.03.0 (2.5, 3.6)1000.38 (0.29, 0.50)1000.0034.7 (3.5, 6.2)1000.11 (0.09, 0.15)100SUR1[3H]GBC1.0537 (447, 646)dExtrapolation to 100% inhibition (see “Results”).100dExtrapolation to 100% inhibition (see “Results”).31 (25, 39)−21 ± 1cThe negative sign indicates an increase in binding (to 141 and 121% of control, respectively (cf. Equation 1)).0.0562 (537, 589)dExtrapolation to 100% inhibition (see “Results”).100dExtrapolation to 100% inhibition (see “Results”).1.4 (0.9, 2.0)22 ± 2Parameters of inhibition curves (K i, inhibition constant; A, amplitude (extent) of curve) were determined from individual binding experiments as described in the legends to Figs. 5 and 7. Hill coefficients were not different from 1.a Percent