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. The of buffer A, specifically bound proteins were eluted with 15 semilogarithmic representation of these results is linear, as ml of a buffer containing 0.1% digitonin, 150 mM NaCl, 150 expected for a pseudo first-order reaction. The apparent rate mM N-acetyl-D-glucosamine, 1 mM IAA, 1 mM PMSF, and constant of association is k = k1([3H]glibenclamide) + kL1, 40 mM imidazole hydrochloride (pH 6.5). Active fractions (2 where k1 and k-1 are the second-order rate constant of ml each) were pooled and loaded onto a 1-ml column made up association and the first-order rate constant of dissociation of of a mixture ofAMP-agarose/GMP-agarose/HA-Ultrogel, 1: the [3H]glibenclamide receptor complex, respectively. The 1:1 (wt/wt), equilibrated in buffer A. The breakthrough was dissociation of [3H]glibenclamide from the sulfonylurea re- collected and retained for [3H]glibenclamide assays and ceptor complex in the detergent extract was measured in the measurements of protein concentrations (12) using bovine presence of an excess of unlabeled glibenclamide. It follows serum albumin as a standard. first-order kinetics (Fig. 1B Inset b). Calculated kinetic Gel electrophoreses were performed using a 4-12% con- constants are k1 = 7.2 x 105 M-1*s-1 and kL1 = 2.2 x 10-4 tinuous gradient polyacrylamide gel (13). Protein samples s 1. The dissociation constant calculated from the kinetic were denatured for 20 min at 56°C in a 75 mM Tris HCl buffer data is Kd = kL1/k, = 0.3 nM. (pH 6.8) containing 2% NaDodSO4, 7.5% glycerol, and 2% Different unlabeled sulfonylureas have been tested for 2-mercaptoethanol. 2-Mercaptoethanol was omitted in non- their ability to interfere with [3H]glibenclamide binding to reducing conditions. Gels were stained with Coomassie blue intact and solubilized binding component (Fig. 2A). The rank or silver (14). order of potency of the different sulfonylureas in inhibiting Photoaffinity Labeling. The intact or solubilized micro- [3H]glibenclamide binding is glibenclamide (Kd = 0.7 nM) > somes were incubated for 1 hr at 4°C with [3H]glibenclamide glipizide (Kd = 2 nM) > tolbutamide (Kd = 7000 nM) > (1-4 nM) in a 1-ml solution containing 150 mM KCI, 40 mM carbutamide (Kd = 15,000 nM). imidazole hydrochloride (pH 6.5), 1 mM IAA, and 0.1 mM An excellent correlation was observed between affinities of PMSF. A parallel incubation was performed in the presence different sulfonylureas for their receptors in intact and of unlabeled glibenclamide (0.1-0.4 ,uM) to measure nonspe- solubilized brain microsomes on one hand and affinities ofthe cific labeling. The samples were irradiated by high-intensity same sulfonylureas for insulinoma cell (RINm5F) micro- UV light with a 2000-W mercury lamp (Philips HP 2000). somes on the other hand (Fig. 2B). Exposure was at 4°C for 8 s at a distance of 20 cm from the Purification of the [3H]Glibenclamide-Binding Activity from lamp. Samples were denatured and analyzed using 4-12% Pig Brain Membranes. The solubilized glibenclamide-binding polyacrylamide gel electrophoresis. After Coomassie blue protein was purified by a combination of four steps: (i) a staining, the gel was treated for fluorography (Amplify chromatography on an HA-Ultrogel column, (ii) an affinity Amersham) and dried. Gels were exposed to Kodak X-Omat chromatography on an ADP-agarose type 4 column, (iii) AR-5 film with a DuPont Cronex intensifying screen for 15- another affinity chromatography on a WGA-Affi-Gel column, 60 days at -70°C. and (iv) a final chromatography on a mixture of AMP- agarose/GMP-agarose/HA-Ultrogel. RESULTS The combination ofthese techniques resulted in a 2500-fold purification from the detergent extract to a final specific Solubilization of the [3H]Glibenclamide-Binding Sites. activity of 1000 pmol/mg ofprotein for the best fraction when Among a number of detergents assayed for brain microsomes starting from the best microsomal preparation. Downloaded by guest on September 28, 2021 9818 Neurobiology: Bernardi et al. Proc. Natl. Acad. Sci. USA 85 (1988)

0 1-1 1c

..E 400 10 A DD i0 200 400 m 0I OmE

m_200 _ X1 CD

0.

h-

0 2 I~'. [3 H] glibenclamide free, nM ~cu B Carbutamide 5 Tolbutamide ~0Q. 6 Gliclazide @@400 0 40 80 Time~100 W HB 699 7 Glibornuride 3,E20~~00 . ,, C) Glisoxepide 2c 8

0 9 G ypkiuionA Glipizide Glibenclamide I I I 10 1 e I I 0 9 8 7 6 5 -log [Kd], M - Brain receptor

FIG. 2. Inhibition of [3H]glibenclamide binding by different sulfonylureas. (A) Competition between [3H]glibenclamide (1 nM) and other unlabeled sulfonylureas for binding to the. solubilized

receptor. *, Glibenclamide; o, glipizide; m, tolbutamide; and *, carbutamide. Nonspecific binding represented 5% of total binding solubilized microsomes, and kinetic data. (A) Equilibrium [3H]gliben- (not shown). The true Kd value is given by K0.5 = Kd{1 + clamide binding to pig brain microsomes. 0, Total binding; [[3H]glibenclamnide]/Kd([3H]glibenclaniide)}, where [[3H]gliben- nonspecific binding. (Inset) Scatchard plot for the specific [3H]- clamide] is the concentration of [3H]glibenclamide used in the glibenclamide binding. (B) Binding of [3H]glibenclamide to solubi- experiment, and Kd([3H]glibenclamide) is the equilibrium dissocia- lized pig brain microsomes. o, Total binding;@*, nonspecific binding. tion constant of the, [3H]glibenclamide receptor complex. (B) Cor-

(Insets a and b) Association and dissociation kinetics for th idng relation curve. Kd values of different hypoglycemic drugs for intact of [3Hlglibenclamide to its solubilized receptor. (Inset a) Association (A) or solubilized (0) pig.brain microsomes were plotted against Kd kinetics for [3H]glibenclamide (1 nM) binding to its solubilized values (taken from ref. 7) of different sulfonylureas for binding to receptor at 40C: time course (0) and pseudo first-order representation insulinoma cell (RINmSF) microsomes (slope = 1.05; r = 0.98). of the data (e). The concentration of free [3H]glibenclamide varied only by 9% during the course of kinetic studies. (Inset b) Dissociation from the WGA column. It indicates a Kd value of 0.8 0.3 kinetics corresponding to specific binding of [3H]glibenclamide (0) nM for the [3H]glibenclamide-receptor complex and a Bmax and first-order representation of the data (e). X, %t maximal [3H1- value of 750 pmol/mg of protein. This latter value corre- glibenclamide bound to the solubilized receptor. (Inset c) Scatchard sponds to a 50-fold purification for the peak fraction. The plot for the specific [3H]glibenclamide binding. B/F, bound/free. [3H]glibenclamide receptor was not specifically retained on Left ordinates in Insets- a and. b correspond to specific [3H]gliben- clamide bound (%o) (0) and right ordinates are as follows: Inset a, other lectins, such as concanavalin A, lectins from Bandeira log[(100 - X (%))] (e); Inset b, log [X (%)] (0). simplicifolia, Asparagus pea, Helix pomatia, Phytollaca americana, or lentil and soybean lectins. In the first step of the purification procedure, solubilized The peak fraction from the WGA column was finally microsomes were loaded on an HA-Ultrogel column. The loaded onto a column containing a mixture of AMP- solubilized [3H]glibenclamide-binding component migrated agarose/GMP-agarose/HA-Ultrogel. Contaminating pro- in the breakthrough with a yield of 85% 5%o (purification teins were adsorbed on this column but the [3H]glibenclam- factor, 2-3). The active breakthrough was then loaded onto ide-binding component was not retained and was recovered an ADP-agarose column. In the absence of ATP, 85% of the with a 100% yield (1.5-fold purification). The total purifica- [3H]glibenclamide-binding component was retained by this tion yield of [3H]glibenclamide-binding sites from the solu- column. Elution was achieved by using buffer A supple- bilized material was 13% 2%. mented with 3 mM ATP (Fig. 3A). Equilibrium binding Fig. 4 shows the NaDodSO4/polyacrylamide gel electro- studies using this purified material indicated a Kd value of 0.8 phoresis of the fraction coming from the last step of purifi- 0.3 nM for the [3H]glibenclamide-reseptor complex and a cation. A single major component ofMr 150,000 ± 10,000 was Bmax value of 15 .pmol/mg of protein for the best fraction, identified under reducing and nonreducing conditions. which corresponded to a 15-fold purification (Fig. 3A Inset). Photoaffinity Labeling of Pig Brain Microsomes, Solubilized The third purification step made use of a column of WGA Microsomes, and Purified Receptor with [3ll]Glibenclamide. coupled to Affi-Gel 10. Retention of 80% ± 5% of the applied The high affinity and the very chemical nature ofthe structure receptor was observed after loading a 10-ml WGA-Affi-Gel ofthe sulfonylurea suggested that [3H]glibenclamide could be column. Fig. 3B presents a typical elution profile from the a very good probe for direct photoaffinity labeling experi- WGA column. Fig. 3B Inset shows the Scatchard plot for ments such as those that have been used for the voltage- specific [3H]glibenclamide binding to the purified material sensitive Ca2l channel using other types of ligands (18). Downloaded by guest on September 28, 2021 Neurobiology: Bemardi et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9819 A B C D E F G H A 2077 11 T-.-I. T -

. 0 5 -lo 150- 0~ ~ ~ ~ ~~

_~ :I_ C.). .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 0 ~ ~ ~ 5 ca5 0 15 Front - _~~~~~~~~~~~~~~~~~~~~~~~4 FIG. 5. Autoradiographic pattern ofphotoaffinity labeling carried Fraction -1~ ~~~0 out with active sulfonylurea-binding fractions eluted from WGA- Affi-Gel (lanes A-D) and ADP-agarose (lanes E-H),. columns with [3H]glibenclamide (3 nM) in the absence (lanes E, and G), and ~~~0 ~ 5 B,P, a 10 B presence (lanes A, C, F, and H) of 0.3 ,uM gliber*lamide. Lanes C, D, G, and H, treated with 2-mercaptoethanol; lanes A, B, E, and F, X 5 5Bnmo/mg 4 without 2-mercaptoethanol. The Mr 150,000 polypeptide is indicated (as Mr X 10-3). was specifically incorporated into a polypeptide of Mr 0 0.5 150,000 ± 5000 (Fig. 5). Gel patterns were identical in the presence and absence of 2-mercaptoethanol.

DISCUSSION Sulfonylurea receptors are present in relatively large amounts in insulinoma cells (150 fmol/mg ofprotein); in these cells the receptors have clearly been shown to be associated Fraction with ATP-regulated K+ channels (7). Sulfonylurea receptors FIG. 3. Purification of the (3H]glibenclamide receptor on ADP- are also present in other cell types, such as cardiac cells (8) agarose type 4 and WGA-Affi-Gel column chromatographies. (A) or neuronal cells (9, 19, 20). Pig brain cortex membranes Elution profile of the specific binding for [3H]glibenclamide (0) and contain a large amount of sulfonylurea receptors (=400 protein concentration (e). (Inset) Scatchard plot for the specific fmol/mg of protein) and have actually a higher binding [3H]glibenclamide binding to ADP-agarose-purified material. (B) Affinity chromatography on WGA column of the [3H]glibenclamide- capacity for this category of drugs than insulinoma cells binding component previously purified on ADP-agarose. (Inset) themselves (150 fmol/mg of protein) (7). Structure-function Scatchiard plot for the specific [3H]glibenclamide binding to WGA- relationships in the sulfonylurea series are the same for purified material. B/F, bound/free. [3HlGlibenclamide was at 1 nM. membranes of insulinoma cells and brain membranes (Fig. 2B). Photoaffinity labeling was performed on intact and solubi- Sulfonylurea-binding sites in brain membranes have not lized membranes and on purified receptor preparations. yet been related with functional neuronal ATP-regulated K+ Typical results presented in Fig. 5 indicate that radioactivity channels. However, because the pharmacological properties of sulfonylurea-binding sites seem to be the same in neuronal 2 cells as in insulin-secreting cells and cardiac cells in which sulfonylurea receptors have been associated with ATP- regulated K+ channels, it seems likely that, also in this type 200 of cell, sulfonylurea receptors will be associated with ATP- regulated K+ channels. The physiological function of the 116 ATP-regulated K+ channel in brain cells remains to be elucidated. However, this channel may play an important 92 role in ionic disorders associated with ischemia in the brain. 66 _ The first step following ischemia corresponds to an elevation of the interstitial K+ concentration in the brain, indicating that a K+ effiux pathway has been opened (21). This intra- 45 _ cellular K+ loss may well be a direct consequence of the decrease ofthe intracellular concentration ofATP linked to the ischemic situation. A good candidate as a transport system responsible for the K+ loss would then be the ATP-regulated FIG. 4. NaDodSO4/polyacrylamide gel electrophoresis of the K+ channel, which is known to be in an open state at low purified [3H]glibenclamide receptor. Electrophoresis was carried out intracellular ATP concentrations. Such a view finds support in in 4-12% polyacrylamide gels under nonreducing and reducing the fact that the initial phase of the K+ leak is slower in conditions. Standards were myosin (Mr 200,000), 8-galactosidase ischemic animals put in hyperglycemic situations (i.e., pre- (Mr 116,000), phosphorylase b (Mr 97,000), bovine serum albumin sumably with higher levels of intracellular ATP) and acceler- (MW 66,000), ovalbumin (M, 45,000), carbonic anhydrase (Mr 31,000), ated in ischemic animals in hypoglycemic situation (21). Other and STI (Mr 21,000). Molecular weights are shown as Mr X 10-3. The Mr 150,000 polypeptide is indicated. Lane 1, active peak from the functions could be ascribed to ATP-regulated K+ channels in WGA fraction after a subsequent chromatography through a mixture the brain, particularly in glucose-sensing hypothalamic neu- of AMP-agarose/GMP-agarose/HA-Ultrogel. Lane 1, treated with rones in charge offeeding regulation (22). These neurones may 2-mercaptoethanol; lane 2, same purified material without 2- emit electrical signals upon glucose perfusion in a way similar mercaptoethanol treatment. Lanes 1 and 2 were silver stained. to beta cells in the pancreas. Downloaded by guest on September 28, 2021 9820 Neurobiology: Bernardi et al. Proc. Natl. Acad. Sci. USA 85 (1988) This paper reports the extensive purification of brain It will be particularly interesting to see whether this type of sulfonylurea-binding sites (2500-fold). The strategy for this channel protein, in spite of the differences in size with other purification has used the idea that (i) this receptor, if it is purified channels, also belongs to the superfamily of channel indeed associated with an ATP-regulated K+ channel, will be structures that already includes voltage-sensitive Na', Ca2+, retained on an ADP-agarose affinity column since it is known and K+ channels (32). (23-26) that ADP itself regulates the activity of this type of channel, (ii) the binding protein, as other binding proteins for We are grateful to Hoechst-Roussel Pharmaceutical Inc. for toxins or cardiovascular drugs or diuretics that alter the generous gifts of [3H]glibenclamide, glibenclamide, HB699, and tolbutamide and to Boehringer Ingelheim, Laboratoires Roche, function of other types of channels, is a glycoprotein and will Shering-Plough Corp., and Laboratoires Servier for gliquidone, therefore be retained on a lectin column, (iii) the receptor will glibornuride, glisoxepide, carbutamide, and gliclazide. We thank Dr. not be retained on AMP-agarose or GMP-agarose columns J. Kitabgi for gifts of chlorpropamide and glipizide and Dr. A. because these mononucleotides are known to be without Lombet for fruitful discussions. Thanks are due to M. Tomkowiak effect on the activity ofthe ATP-regulated K+ channel in beta and C. Roulinat-Bettelheim for expert technical assistance. This cells and cardiac cells (23-25). work was supported by the Centre National de la Recherche The solubilized receptor is analogous in its pharmacolog- Scientifique, the Fondation pour la Recherche Mddicale, and the ical properties to the membrane receptor. It has a similar Mutuelle Gdndrale de l'Education Nationale. affinity (Kd = 0.8 + 0.3 nM) for glibenclamide and a similar rank order for the recognition of different drugs in the 1. Petersen, 0. & Findlay, I. (1987) Physiol. Rev. 67, 1054-1116. 2. Stanfield, P. R. (1987) Trends Neurosci. 10, 335-339. sulfonylurea series. 3. Ashcroft, F. M. (1988) Annu. Rev. Neurosci. 11, 97-118. The purified sulfonylurea-binding component appears as a 4. Loubatieres, A. (1977) in The Diabetic Pancreas, eds. Volk, single band with a Mr of 150,000 ± 10,000. The mobility ofthis B. W. & Wellmann, K. E. (Bailliere Tindall, London), pp. 489- band is unaltered by reduction. If the stoichiometry is of one 515. sulfonylurea-binding site per binding component of Mr 5. Sturgess, N. C., Ashford, M. L. J., Cook, D. L. & Hales, 150,000, one can calculate that the maximal binding capacity C. N. (1985) Lancet ii, 474-475. of the pure receptor should be 6.7 nmol/mg of protein. Our 6. Schmid-Antomarchi, H., De Weille, J. R., Fosset, M. & best preparations only had a specific activity of 1 nmol/mg of Lazdunski, M. (1987) Biochem. Biophys. Res. Commun. 146, protein. Several explanations can be given for the fact that 21-25. the 7. Schmid-Antomarchi, H., De Weille, J. R., Fosset, M. & the specific activity is not high enough whereas gel Lazdunski, M. (1987) J. Biol. Chem. 262, 15840-15844. pattern seems to be very satisfactory. The first one is of 8. Fosset, M., De Weille, J. R., Green, R. D., Schmid-Anto- course that the putative ATP-regulated K+ channel is an marchi, H. & Lazdunski, M. (1988) J. Biol. Chem. 263, oligomeric protein and that the stoichiometry is of less than 7933-7936. one glibenclamide-binding site per channel protein. The most 9. Geisen, K., Hitzel, V., Okomonopoulos, R., Puinter, J., Weyer, likely one is that there is an underestimation of the final R. & Summ, H. D. (1985) Arzneim.-Forsch. 35, 707-712. binding capacity, which is probably due to the relatively short 10. Gaines, K. L., Hamilton, S. & Boyd, A. E., III (1988) J. Biol. half-life of the purified receptor (12 hr at 40C). Since it takes Chem. 263, 2589-2592. about that of time to the 11. Krueger, B. K., Ratzlaff, R. W., Strichartz, G. R. & Blaustein, length purify sulfonylurea-binding M. D. (1979) J. Membr. Biol. 50, 287-310. protein, then one should never expect an activity better than 12. Peterson, G. I. (1977) Anal. Biochem. 83, 346-356. about 3 nmol/mg of protein. The protein with a Mr of 150,000 13. Laemmli, U. K. (1970) Nature (London) 227, 680-685. is probably a mixture of active and inactivated binding com- 14. Merril, C. R., Goldman, D., Sedman, S. A. & Ebert, M. H. ponents. Previous purifications of voltage-dependent Na' (1981) Science 211, 1437-1438. and Ca.2 channels (15-17) have also led to final specific 15. Levinson, S. R., Duch, D. S., Urban, B. W. & Recio-Pinto, E. activities that, for the same reasons, were well under the (1986) Ann. N. Y. Acad. Sci. 479, 162-178. expected values. 16. Lombet, A. & Lazdunski, M. (1984) Eur. J. Biochem. 141, 651- One very convincing aspect of the results is that the 660. of the com- 17. Borsotto, M., Norman, R. I., Fosset, M. & Lazdunski, M. molecular weight purified sulfonylurea-binding (1984) Eur. J. Biochem. 142, 449-455. ponent is very similar if not identical to the molecular weight 18. Galizzi, J.-P., Borsotto, M., Barhanin, J., Fosset, M. & of the protein band that has been identified as the sulfonyl- Lazdunski, M. (1986) J. Biol. Chem. 261, 1393-1397. urea receptor by direct affinity labeling with [3H]gliben- 19. Kaubisch, N., Hammer, R., Wollheim, C., Renold, A. E. & clamide (Fig. 5). The same type of affinity labeling has Offord, R. E. (1982) Biochem. Pharmacol. 31, 1171-1174. recently been independently tried on insulinoma cells. The 20. Lupo, B. & Bataille, D. (1987) Eur. J. Pharmacol. 140, 157-169. labeled band identified after gel slicing (instead of autoradi- 21. Hansen, A. J. (1985) Physiol. Rev. 65, 101-148. ography as shown in Fig. 5) has suggested. a Mr of 140,000 22. Minami, T., Oomura, Y. & Sugimori, M. (1986) J. Physiol. 380, (27). The similarities of the molecular weights found with 127-143. beta of 23. Noma, A. (1983) Nature (London) 305, 147-148. brain- and cell-binding components sulfonylureas 24. Kakei, M. Kelly, R. P., Ashcroft, S. J. H. & Ashcroft, F. M. provide another indication that in both cases they correspond (1986) FEBS Lett. 208, 63-66. to ATP-regulated K+ channels. 25. Dunne, M. J. & Petersen, 0. H. (1986) FEBS Lett. 208, 59-62. The subunit constitution of the sulfonylurea receptors 26. Misler, S., Falke, L. C., Gillis, K. & McDaniel, M. L. (1986) seems to be different from that found for other identified K+ Proc. Natl. Acad. Sci. USA 83, 7119-7123. channels. A family of voltage-dependent K+ channels has 27. Kramer, W., Oekonomopulos, R., Punter, J. & Summ, H.-D. recently been cloned by using the Shaker mutation in Dro- (1988) FEBS Lett. 229, 355-359. sophila. Drosophila K channels of the A type have been 28. Timpe, L. C., Schwarz, T. L., Tempel, B. L., Papazian, found to have a Mr of 70,000 (28). Interestingly, the size of D. M., Jan, Y. N. & Jan, L. Y. (1988) Nature (London) 331, is same as that found for 143-145. this channel protein the voltage- 29. Black, A. R. & Dolly, J. 0. (1986) Eur. J. Biochem. 156, 609- sensitive K+ channels identified in mammalian brain with two 617. specific toxins-i.e., dendrotoxin (29-31) and mast cell 30. Rehm, H., Bidard, J.-N., Schweitz, H. & Lazdunski, M. (1988) degranulating peptide (30, 31). Biochemistry 21, 1827-1832. The purification and elucidation of the subunit structure of 31. Rehm, H. & Lazdunski, M. (1988) Proc. Natl. Acad. Sci. USA the ATP-regulated K+ channel open the way for a molecular 85, 4919-4923. analysis of the ATP-regulated K+ channel at the DNA level. 32. Stevens, C. F. (1987) Nature (London) 328, 198-199. Downloaded by guest on September 28, 2021