Proc. Natl. Acad. Sci. USA Vol. 85, pp. 9816-9820, December 1988 Neurobiology Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel (sulfonylurea/ischemia) HENRI BERNARDI, MICHEL FOSSET, AND MICHEL LAZDUNSKI* Centre de Biochimie, Centre National de la Recherche Scientifique, Parc Valrose, 06034 Nice Cedex, France Communicated by Jean-Marie Lehn, September 7, 1988 (received for review May 6, 1988) ABSTRACT Sulfonylurea and particularly glibenclamide ATP, and wheat germ agglutinin (WGA) were purchased are potent blockers of ATP-regulated K+ channels in insulin- from Sigma. ADP-agarose type 4 (ADP attached through the secreting cells. A very good correlation exists between binding ribose hydroxyls by means of periodate oxidation), AMP- of sulfonylurea to brain and insulinoma cell membranes. The agarose, and GMP-agarose gels were from Pharmacia. Hy- [3H]glibenclamide-binding component from pig brain micro- droxylapatite (HA-Ultrogel) was from IBF (Villeneuve- somes was solubilized with digitonin with a complete retention la-Garenne, France). Pig brains were collected at the local of its properties of interaction with glibenclamide and other slaughterhouse 30 min after death and immediately stored in sulfonylureas. A four-step purification was achieved that used liquid nitrogen until processed. (i) hydroxylapatite chromatography, (ii and iii) affinity chro- Microsome Preparation and Solubilization of [3H]Gliben- matographies on ADP-agarose and wheat germ agglutinin- clamide-Binding Sites. Microsomes were prepared from pig agarose columns, and (iv) a rmal chromatographic step on a brain cortex in 40 mM imidazole hydrochloride buffer (pH mixture of AMP-agarose/GMP-agarose/hydroxylapatite. This 6.5) in the presence of a mixture of protease inhibitors [1 mM procedure led to a 2500-fold purification. NaDodSO4/poly- EDTA/1 mM iodoacetamide (IAA)/0.1 mM phenylmethyl- acrylamide gel electrophoresis of the purified material in reduc- sulfonyl fluoride (PMSF)/10 gg of soybean trypsin inhibitor ing and nonreducing conditions showed that the sulfonylurea- (STI) per ml/10 ,M leupeptin]. Fifteen grams of pig cortex binding component is made of a single major polypeptide chain was homogenized in 500 ml of buffer at 40C with a Polytron of Mr 150,000 ± 10,000. Direct photoafmnity labeling of the PT10 homogenizer (Kinematica GmbH, Lucerne, Switzer- receptor with [3H]glibenclamide at different steps of the purifi- land) (setting 6, 1 min) and centrifuged as described (11) to cation also showed that radioactivity was specifically incorpo- obtain microsomes. rated into a polypeptide ofMr 150,000 ± 5000, thus confirming Pig brain microsomes were pelleted at 80,000 x g for 20 min the subunit structure indicated by the purification. and resuspended in the solubilization buffer (1.8% digitonin/ 100 mM KCI/ 40 mM imidazole hydrochloride, pH 6.5/1 mM ATP-sensitive K+ channels have recently been identified in IAA/0.1 mM PMSF/10 ,ug of STI per ml/10 ,uM leupeptin) pancreatic beta cells, cardiac cells, and skeletal muscle cells to give a final concentration of 5-8 mg of protein per ml. The (1-3). Their physiological role is best understood in pancre- solution was agitated for 1 hr at 4°C and centrifuged at 135,000 atic beta cells, in which their blockade in response to glucose x g for 20 min. The supernatant was then collected and stored perfusion involves a depolarization that triggers repetitive at 4°C for a maximum of 24 hr. electrical activity that subsequently provokes Ca2" entry and (3HjGlibenclamide-Binding Assay. For equilibrium binding insulin secretion (3). studies, intact and solubilized microsomes (500 ,g/ml) were Sulfonylureas are hypoglycemic agents that have been incubated in the presence of increasing concentrations of used for a long time in the treatment of diabetes mellitus (4). [3H]glibenclamide for 1 hr at 4°C. The assay medium was It is now well established that molecules in this family of buffered with 20 mM imidazole hydrochloride (pH 6.5). drugs are specific blockers of ATP-regulated K+ channels in When working with intact microsomes, bound and free insulin-secreting cells (5-7) and cardiac cells (8). ligands were separated by filtration of aliquots on GF/C Binding sites for [3H]glibenclamide, the most potent sul- Whatman filters. Filters were rapidly washed twice with 6 ml fonylurea (6-8), have now been identified in pancreatic beta of ice-cold 100 mM Tris HCI (pH 7.5) and radioactivity was cells (7, 9, 10), cardiac cells (8), and brain membranes (9). counted in Biofluor liquid scintillation fluid (New England This paper reports the identification by purification and by Nuclear). For binding studies with solubilized microsomes, photoaffinity labeling of the subunit structure of the [3H]- samples of 200 ,l were loaded onto 5 ml of Sephadex G-50 glibenclamide-binding protein from pig brain cortex, which is medium columns equilibrated with 0.05% 3-[(3-cholamido- presumably associated with a neuronal ATP-regulated K+ propyl)dimethylammonio]-1-propanesulfonate/20 mM imi- channel. dazole hydrochloride, pH 6.5. The bound radioactivity was eluted by 2.4 ml of0.5 M NaCl and fractions were assayed for MATERIALS AND METHODS radioactivity as described above. Specific [3H]glibenclamide = binding was calculated by subtraction from the total binding Materials. [3H]Glibenclamide (50 Ci/mmol; 1 Ci 37 GBq) ofthe nonspecific binding component determined in a parallel was from Hoechst. Clorpropamide, HB699, tolbutamide, unlabeled 1 carbutamide, gliclazide, glibornuride, glipizide, glisoxepide, incubation with ,M glibenclamide. gliquidone, and glibenclamide were generously provided by Roche, Schering, Servier, and Pfizer. Digitonin, Abbreviations: ADP-agarose type 4 column, ADP attached through Hoechst, the ribose hydroxyls by means of periodate oxidation; HA-Ultrogel, hydroxylapatite; IAA, iodoacetamide; PMSF, phenylmethylsulfonyl The publication costs of this article were defrayed in part by page charge fluoride; STI, soybean trypsin inhibitor; WGA, wheat germ agglu- payment. This article must therefore be hereby marked "advertisement" tinin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be sent. 9816 Downloaded by guest on September 28, 2021 Neurobiology: Bernardi et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9817 In competition experiments between [3H]glibenclamide solubilization, only digitonin, Nonidet P-40, and 3-[3-cholami- and unlabeled sulfonylureas, intact or solubilized micro- dopropyl)dimethylammonio]-1-propanesulfonate were able to somes were incubated for 1 hr at 4TC in the presence of 1 nM solubilize the [3H]glibenclamide-binding component with a [3H]glibenclamide and of various concentrations of the un- high yield. Other detergents, such as cholate, desoxycholate, labeled compounds. Lubrol PX, Tween 20, Triton X-100, Emulphogen BC720, Association and Dissociation Kinetics. Kinetics of associa- and Empigen BB/P, only solubilized the [3H]glibenclamide tion of [3H]glibenclamide to its solubilized receptor were receptor but with a very low efficiency. Solubilization with measured with 1 nM [3H]glibenclamide added at time zero. Nonidet P-40 produced a labile receptor (ti/2 = 2 hr) that could Aliquots were taken at different times and the bound radio- not be stabilized by adding phospholipids or glycerol. Digi- activity was measured by the techniques described above. tonin was the only detergent that allowed adequate solubili- When the level of specifically bound [3H]glibenclamide had zation permitting purification of the [3H]glibenclamide-bind- reached a plateau value, dissociation was started by adding ing component. The most successful solubilization of the a large excess (5 /iM) of glibenclamide. [3H]glibenclamide receptor was obtained with 1.8% digito- Chromatographies on HA-Ultrogel, ADP-Agarose, WGA- nin, giving a yield of 40% ± 5% of active receptor. High Affi-Gel, AMP-Agarose, and GMP-Agarose Columns. HA- concentrations of KCI (up to 1 M), NaCl (up to 2 M), or Ultrogel columns (40 ml) were equilibrated in 0.05% digitonin (up to 2%) did not interfere with [3H]glibenclamide digitonin/100 mM KCI/40 mM imidazole hydrochloride, pH binding. In contrast to observations made in the course of 6.5/1 mM IAA/0.1 mM PMSF. Solubilized microsomes (70 purification ofthe Na' channel (15, 16) or the skeletal muscle ml, 175 mg of protein) were loaded on the column and eluted Ca2+ channel (17), the addition of phospholipids and/or with the same buffer. The eluate was adjusted to a final glycerol failed to increase the of the concentration of 150 mM KCl/1 mM EDTA and applied to stability sulfonylurea the ADP-agarose column (15 ml) equilibrated in buffer A receptor. (0.1% digitonin/150 mM KCI/40 mM imidazole hydrochlo- [3H]Glibenclamide equilibrium binding studies indicate ride, pH 6.5/1 mM IAA/0.1 mM PMSF). The column was that this sulfonylurea specifically binds to a single class of first washed with 30 ml of the equilibration buffer and then noninteracting sites in intact and solubilized microsomes washed with 30 ml of a buffer containing 0.1% digitonin, 300 (Fig. 1 A and B). The apparent equilibrium dissociation mM NaCl, 1 mM IAA, 0.1 mM PMSF, and 40 mM imidazole constants (Kd) and the maximal binding capacities (Bmax) hydrochloride (pH 6.5). Elution was carried out with buffer relative to [3H]glibenclamide interaction were identical for A containing 3 mM ATP. Five-milliliter fractions were intact and solubilized microsomes: Kd = 0.8 + 0.3 nM and collected and assayed for [3H]glibenclamide binding and Bmax = 400 ± 50 fmol/mg (Fig. 1 A Inset and B Inset c). protein concentration. Active fractions were pooled and Association and Dissociation Kinetics. Typical kinetics of recycled for 2 hr on a WGA-Affi-Gel column (10 ml) that was association of [3H]glibenclamide to detergent extracts of equilibrated overnight in buffer A. After washing with 40 ml brain microsomes are presented in Fig. 1B Insets a and b.