Proc. Natl. Acad. Sci. USAVol. 85, pp. 9816-9820, December 1988Neurobiology

Characterization, purification, and affinity labeling of the brain[3H]glibenclamide-binding protein, a putative neuronalATP-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 glibenclamideare potent blockers of ATP-regulated K+ channels in insulin-secreting cells. A very good correlation exists between bindingof sulfonylurea to brain and insulinoma cell membranes. The[3H]glibenclamide-binding component from pig brain micro-somes was solubilized with digitonin with a complete retentionof its properties of interaction with glibenclamide and othersulfonylureas. A four-step purification was achieved that used(i) hydroxylapatite chromatography, (ii and iii) affinity chro-matographies on ADP-agarose and wheat germ agglutinin-agarose columns, and (iv) a rmal chromatographic step on amixture of AMP-agarose/GMP-agarose/hydroxylapatite. Thisprocedure led to a 2500-fold purification. NaDodSO4/poly-acrylamide gel electrophoresis of the purified material in reduc-ing and nonreducing conditions showed that the sulfonylurea-binding component is made of a single major polypeptide chainof Mr 150,000 ± 10,000. Direct photoafmnity labeling of thereceptor with [3H]glibenclamide at different steps of the purifi-cation also showed that radioactivity was specifically incorpo-rated into a polypeptide of Mr 150,000 ± 5000, thus confirmingthe subunit structure indicated by the purification.

ATP-sensitive K+ channels have recently been identified inpancreatic beta cells, cardiac cells, and skeletal muscle cells(1-3). Their physiological role is best understood in pancre-atic beta cells, in which their blockade in response to glucoseperfusion involves a depolarization that triggers repetitiveelectrical activity that subsequently provokes Ca2" entry andinsulin secretion (3).

Sulfonylureas are hypoglycemic agents that have beenused for a long time in the treatment of diabetes mellitus (4).It is now well established that molecules in this family ofdrugs are specific blockers of ATP-regulated K+ channels ininsulin-secreting cells (5-7) and cardiac cells (8).

Binding sites for [3H]glibenclamide, the most potent sul-fonylurea (6-8), have now been identified in pancreatic betacells (7, 9, 10), cardiac cells (8), and brain membranes (9).

This paper reports the identification by purification and byphotoaffinity labeling of the subunit structure of the [3H]-glibenclamide-binding protein from pig brain cortex, which ispresumably associated with a neuronal ATP-regulated K+channel.

MATERIALS AND METHODSMaterials. [3H]Glibenclamide (50 Ci/mmol; 1 Ci = 37 GBq)

was from Hoechst. Clorpropamide, HB699, tolbutamide,carbutamide, gliclazide, glibornuride, glipizide, glisoxepide,gliquidone, and glibenclamide were generously provided byHoechst, Roche, Schering, Servier, and Pfizer. Digitonin,

ATP, and wheat germ agglutinin (WGA) were purchasedfrom Sigma. ADP-agarose type 4 (ADP attached through theribose hydroxyls by means of periodate oxidation), AMP-agarose, and GMP-agarose gels were from Pharmacia. Hy-droxylapatite (HA-Ultrogel) was from IBF (Villeneuve-la-Garenne, France). Pig brains were collected at the localslaughterhouse 30 min after death and immediately stored inliquid nitrogen until processed.Microsome Preparation and Solubilization of [3H]Gliben-

clamide-Binding Sites. Microsomes were prepared from pigbrain cortex in 40 mM imidazole hydrochloride buffer (pH6.5) in the presence of a mixture of protease inhibitors [1 mMEDTA/1 mM iodoacetamide (IAA)/0.1 mM phenylmethyl-sulfonyl fluoride (PMSF)/10 gg of soybean trypsin inhibitor(STI) per ml/10 ,M leupeptin]. Fifteen grams of pig cortexwas homogenized in 500 ml of buffer at 40C with a PolytronPT10 homogenizer (Kinematica GmbH, Lucerne, Switzer-land) (setting 6, 1 min) and centrifuged as described (11) toobtain microsomes.

Pig brain microsomes were pelleted at 80,000 x g for 20 minand resuspended in the solubilization buffer (1.8% digitonin/100 mM KCI/ 40 mM imidazole hydrochloride, pH 6.5/1 mMIAA/0.1 mM PMSF/10 ,ug of STI per ml/10 ,uM leupeptin)to give a final concentration of 5-8 mg of protein per ml. Thesolution was agitated for 1 hr at 4°C and centrifuged at 135,000x g for 20 min. The supernatant was then collected and storedat 4°C for a maximum of 24 hr.

(3HjGlibenclamide-Binding Assay. For equilibrium bindingstudies, intact and solubilized microsomes (500 ,g/ml) wereincubated in the presence of increasing concentrations of[3H]glibenclamide for 1 hr at 4°C. The assay medium wasbuffered with 20 mM imidazole hydrochloride (pH 6.5).When working with intact microsomes, bound and freeligands were separated by filtration of aliquots on GF/CWhatman filters. Filters were rapidly washed twice with 6 mlof ice-cold 100 mM Tris HCI (pH 7.5) and radioactivity wascounted in Biofluor liquid scintillation fluid (New EnglandNuclear). For binding studies with solubilized microsomes,samples of 200 ,l were loaded onto 5 ml of Sephadex G-50medium columns equilibrated with 0.05% 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate/20 mM imi-dazole hydrochloride, pH 6.5. The bound radioactivity waseluted by 2.4 ml of 0.5 M NaCl and fractions were assayed forradioactivity as described above. Specific [3H]glibenclamidebinding was calculated by subtraction from the total bindingofthe nonspecific binding component determined in a parallelincubation with unlabeled 1 ,M glibenclamide.

Abbreviations: ADP-agarose type 4 column, ADP attached throughthe ribose hydroxyls by means of periodate oxidation; HA-Ultrogel,hydroxylapatite; IAA, iodoacetamide; PMSF, phenylmethylsulfonylfluoride; STI, soybean trypsin inhibitor; WGA, wheat germ agglu-tinin.*To whom reprint requests should be sent.

9816

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 85 (1988) 9817

In competition experiments between [3H]glibenclamideand unlabeled sulfonylureas, intact or solubilized micro-somes were incubated for 1 hr at 4TC in the presence of 1 nM[3H]glibenclamide and of various concentrations of the un-labeled compounds.

Association and Dissociation Kinetics. Kinetics of associa-tion of [3H]glibenclamide to its solubilized receptor weremeasured with 1 nM [3H]glibenclamide added at time zero.Aliquots were taken at different times and the bound radio-activity was measured by the techniques described above.When the level of specifically bound [3H]glibenclamide hadreached a plateau value, dissociation was started by addinga large excess (5 /iM) of glibenclamide.

Chromatographies on HA-Ultrogel, ADP-Agarose, WGA-Affi-Gel, AMP-Agarose, and GMP-Agarose Columns. HA-Ultrogel columns (40 ml) were equilibrated in 0.05%digitonin/100 mM KCI/40 mM imidazole hydrochloride, pH6.5/1 mM IAA/0.1 mM PMSF. Solubilized microsomes (70ml, 175 mg of protein) were loaded on the column and elutedwith the same buffer. The eluate was adjusted to a finalconcentration of 150 mM KCl/1 mM EDTA and applied tothe ADP-agarose column (15 ml) equilibrated in buffer A(0.1% digitonin/150 mM KCI/40 mM imidazole hydrochlo-ride, pH 6.5/1 mM IAA/0.1 mM PMSF). The column wasfirst washed with 30 ml of the equilibration buffer and thenwashed with 30 ml of a buffer containing 0.1% digitonin, 300mM NaCl, 1 mM IAA, 0.1 mM PMSF, and 40 mM imidazolehydrochloride (pH 6.5). Elution was carried out with bufferA containing 3 mM ATP. Five-milliliter fractions werecollected and assayed for [3H]glibenclamide binding andprotein concentration. Active fractions were pooled andrecycled for 2 hr on a WGA-Affi-Gel column (10 ml) that wasequilibrated overnight in buffer A. After washing with 40 mlof buffer A, specifically bound proteins were eluted with 15ml of a buffer containing 0.1% digitonin, 150 mM NaCl, 150mM N-acetyl-D-glucosamine, 1 mM IAA, 1 mM PMSF, and40 mM imidazole hydrochloride (pH 6.5). Active fractions (2ml each) were pooled and loaded onto a 1-ml column made upof a mixture of AMP-agarose/GMP-agarose/HA-Ultrogel, 1:1:1 (wt/wt), equilibrated in buffer A. The breakthrough wascollected and retained for [3H]glibenclamide assays andmeasurements of protein concentrations (12) using bovineserum albumin as a standard.Gel electrophoreses were performed using a 4-12% con-

tinuous gradient polyacrylamide gel (13). Protein sampleswere denatured for 20 min at 56°C in a 75 mM Tris HCl buffer(pH 6.8) containing 2% NaDodSO4, 7.5% glycerol, and 2%2-mercaptoethanol. 2-Mercaptoethanol was omitted in non-reducing conditions. Gels were stained with Coomassie blueor silver (14).

Photoaffinity Labeling. The intact or solubilized micro-somes were incubated for 1 hr at 4°C with [3H]glibenclamide(1-4 nM) in a 1-ml solution containing 150 mM KCI, 40 mMimidazole hydrochloride (pH 6.5), 1 mM IAA, and 0.1 mMPMSF. A parallel incubation was performed in the presenceof unlabeled glibenclamide (0.1-0.4 ,uM) to measure nonspe-cific labeling. The samples were irradiated by high-intensityUV light with a 2000-W mercury lamp (Philips HP 2000).Exposure was at 4°C for 8 s at a distance of 20 cm from thelamp. Samples were denatured and analyzed using 4-12%polyacrylamide gel electrophoresis. After Coomassie bluestaining, the gel was treated for fluorography (AmplifyAmersham) and dried. Gels were exposed to Kodak X-OmatAR-5 film with a DuPont Cronex intensifying screen for 15-60 days at -70°C.

RESULTSSolubilization of the [3H]Glibenclamide-Binding Sites.

Among a number of detergents assayed for brain microsomes

solubilization, only digitonin, Nonidet P-40, and 3-[3-cholami-dopropyl)dimethylammonio]-1-propanesulfonate were able tosolubilize the [3H]glibenclamide-binding component with ahigh yield. Other detergents, such as cholate, desoxycholate,Lubrol PX, Tween 20, Triton X-100, Emulphogen BC720,and Empigen BB/P, only solubilized the [3H]glibenclamidereceptor but with a very low efficiency. Solubilization withNonidet P-40 produced a labile receptor (ti/2 = 2 hr) that couldnot be stabilized by adding phospholipids or glycerol. Digi-tonin was the only detergent that allowed adequate solubili-zation permitting purification of the [3H]glibenclamide-bind-ing component. The most successful solubilization of the[3H]glibenclamide receptor was obtained with 1.8% digito-nin, giving a yield of 40% ± 5% of active receptor. Highconcentrations of KCI (up to 1 M), NaCl (up to 2 M), ordigitonin (up to 2%) did not interfere with [3H]glibenclamidebinding. In contrast to observations made in the course ofpurification of the Na' channel (15, 16) or the skeletal muscleCa2+ channel (17), the addition of phospholipids and/orglycerol failed to increase the stability of the sulfonylureareceptor.

[3H]Glibenclamide equilibrium binding studies indicatethat this sulfonylurea specifically binds to a single class ofnoninteracting sites in intact and solubilized microsomes(Fig. 1 A and B). The apparent equilibrium dissociationconstants (Kd) and the maximal binding capacities (Bmax)relative to [3H]glibenclamide interaction were identical forintact and solubilized microsomes: Kd = 0.8 + 0.3 nM andBmax = 400 ± 50 fmol/mg (Fig. 1 A Inset and B Inset c).

Association and Dissociation Kinetics. Typical kinetics ofassociation of [3H]glibenclamide to detergent extracts ofbrain microsomes are presented in Fig. 1B Insets a and b. Thesemilogarithmic representation of these results is linear, asexpected for a pseudo first-order reaction. The apparent rateconstant of association is k = k1([3H]glibenclamide) + kL1,where k1 and k-1 are the second-order rate constant ofassociation and the first-order rate constant of dissociation ofthe [3H]glibenclamide receptor complex, respectively. Thedissociation of [3H]glibenclamide from the sulfonylurea re-ceptor complex in the detergent extract was measured in thepresence of an excess of unlabeled glibenclamide. It followsfirst-order kinetics (Fig. 1B Inset b). Calculated kineticconstants are k1 = 7.2 x 105 M-1*s-1 and kL1 = 2.2 x 10-4s 1. The dissociation constant calculated from the kineticdata is Kd = kL1/k, = 0.3 nM.

Different unlabeled sulfonylureas have been tested fortheir ability to interfere with [3H]glibenclamide binding tointact and solubilized binding component (Fig. 2A). The rankorder of potency of the different sulfonylureas in inhibiting[3H]glibenclamide binding is glibenclamide (Kd = 0.7 nM) >glipizide (Kd = 2 nM) > tolbutamide (Kd = 7000 nM) >carbutamide (Kd = 15,000 nM).An excellent correlation was observed between affinities of

different sulfonylureas for their receptors in intact andsolubilized brain microsomes on one hand and affinities ofthesame sulfonylureas for insulinoma cell (RINm5F) micro-somes on the other hand (Fig. 2B).

Purification of the [3H]Glibenclamide-Binding Activity fromPig Brain Membranes. The solubilized glibenclamide-bindingprotein was purified by a combination of four steps: (i) achromatography on an HA-Ultrogel column, (ii) an affinitychromatography on an ADP-agarose type 4 column, (iii)another affinity chromatography on a WGA-Affi-Gel column,and (iv) a final chromatography on a mixture of AMP-agarose/GMP-agarose/HA-Ultrogel .The combination ofthese techniques resulted in a 2500-fold

purification from the detergent extract to a final specificactivity of 1000 pmol/mg of protein for the best fraction whenstarting from the best microsomal preparation.

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9818 Neurobiology: Bernardi et al.

..E 400

i0 200 400

m_200 _ X1

0 2[3 H] glibenclamide free, nM

@@400 0 40 80 Time~100 W

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solubilized microsomes, and kinetic data. (A) Equilibrium [3H]gliben-

clamide binding to pig brain microsomes. 0, Total binding;

nonspecific binding. (Inset) Scatchard plot for the specific [3H]-

glibenclamide binding. (B) Binding of [3H]glibenclamide to solubi-

lized pig brain microsomes. o, Total binding;@*, nonspecific binding.

(Insets a and b) Association and dissociation kinetics for th idngof[3Hlglibenclamide to its solubilized receptor. (Inset a) Association

kinetics for [3H]glibenclamide (1 nM) binding to its solubilized

receptor at 40C: time course (0) and pseudo first-order representation

of the data (e). The concentration of free [3H]glibenclamide varied

only by 9% during the course of kinetic studies. (Inset b) Dissociation

kinetics corresponding to specific binding of [3H]glibenclamide (0)

and first-order representation of the data (e). X, %t maximal [3H1-

glibenclamide bound to the solubilized receptor. (Inset c) Scatchard

plot for the specific [3H]glibenclamide binding. B/F, bound/free.

Left ordinates in Insets- a and. b correspond to specific [3H]gliben-

clamide bound (%o) (0) and right ordinates are as follows: Inset a,

log[(100 - X (%))] (e); Inset b, log [X (%)] (0).

In the first step of the purification procedure, solubilized

microsomes were loaded on an HA-Ultrogel column. The

solubilized [3H]glibenclamide-binding component migrated

in the breakthrough with a yield of 85% 5%o (purification

factor, 2-3). The active breakthrough was then loaded onto

an ADP-agarose column. In the absence of ATP, 85% of the

[3H]glibenclamide-binding component was retained by this

column. Elution was achieved by using buffer A supple-

mented with 3 mM ATP (Fig. 3A). Equilibrium binding

studies using this purified material indicated a Kd value of 0.8

0.3 nM for the [3H]glibenclamide-reseptor complex and a

Bmax value of 15 .pmol/mg of protein for the best fraction,

which corresponded to a 15-fold purification (Fig. 3A Inset).

The third purification step made use of a column of WGA

coupled to Affi-Gel 10. Retention of80% ± 5% of the appliedreceptor was observed after loading a 10-ml WGA-Affi-Gelcolumn. Fig. 3B presents a typical elution profile from theWGA column. Fig. 3B Inset shows the Scatchard plot forspecific [3H]glibenclamide binding to the purified material

0

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6

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FIG. 2. Inhibition of [3H]glibenclamide binding by differentsulfonylureas. (A) Competition between [3H]glibenclamide (1 nM)and other unlabeled sulfonylureas for binding to the. solubilizedreceptor. *, Glibenclamide; o, glipizide; m, tolbutamide; and *,carbutamide. Nonspecific binding represented 5% of total binding(not shown). The true Kd value is given by K0.5 = Kd{1 +[[3H]glibenclamnide]/Kd([3H]glibenclaniide)}, where [[3H]gliben-clamide] is the concentration of [3H]glibenclamide used in theexperiment, and Kd([3H]glibenclamide) is the equilibrium dissocia-tion constant of the, [3H]glibenclamide receptor complex. (B) Cor-relation curve. Kd values of different hypoglycemic drugs for intact(A) or solubilized (0) pig.brain microsomes were plotted against Kdvalues (taken from ref. 7) of different sulfonylureas for binding toinsulinoma cell (RINmSF) microsomes (slope = 1.05; r = 0.98).

from the WGA column. It indicates a Kd value of 0.8 0.3nM for the [3H]glibenclamide-receptor complex and a Bmax

value of 750 pmol/mg of protein. This latter value corre-

sponds to a 50-fold purification for the peak fraction. The[3H]glibenclamide receptor was not specifically retained onother lectins, such as concanavalin A, lectins from Bandeirasimplicifolia, Asparagus pea, Helix pomatia, Phytollacaamericana, or lentil and soybean lectins.The peak fraction from the WGA column was finally

loaded onto a column containing a mixture of AMP-agarose/GMP-agarose/HA-Ultrogel. Contaminating pro-teins were adsorbed on this column but the [3H]glibenclam-ide-binding component was not retained and was recoveredwith a 100% yield (1.5-fold purification). The total purifica-tion yield of [3H]glibenclamide-binding sites from the solu-bilized material was 13% 2%.

Fig. 4 shows the NaDodSO4/polyacrylamide gel electro-phoresis of the fraction coming from the last step of purifi-cation. A single major component ofMr 150,000 ± 10,000 wasidentified under reducing and nonreducing conditions.

Photoaffinity Labeling of Pig Brain Microsomes, SolubilizedMicrosomes, and Purified Receptor with [3ll]Glibenclamide.The high affinity and the very chemical nature ofthe structureofthe sulfonylurea suggested that [3H]glibenclamide could bea very good probe for direct photoaffinity labeling experi-ments such as those that have been used for the voltage-sensitive Ca2l channel using other types of ligands (18).

B CarbutamideTolbutamide

GliclazideHB 699

GlibornurideGlisoxepide

G ypkiuionA GlipizideGlibenclamide

e I I I I I

Proc. Natl. Acad. Sci. USA 85 (1988)

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Proc. Natl. Acad. Sci. USA 85 (1988) 9819

A 2077

. 0 5 -lo0~~ ~ ~ ~~

C.).0 ~ ~ ~ 5

ca5 0 15

Fraction

a 10 B

-1~ ~~~0

~~~0 ~ 5

0 0.5

X 5 5Bnmo/mg 4

Fraction

FIG. 3. Purification of the (3H]glibenclamide receptor on ADP-agarose type 4 and WGA-Affi-Gel column chromatographies. (A)Elution profile of the specific binding for [3H]glibenclamide (0) andprotein concentration (e). (Inset) Scatchard plot for the specific[3H]glibenclamide binding to ADP-agarose-purified material. (B)Affinity chromatography on WGA column of the [3H]glibenclamide-binding component previously purified on ADP-agarose. (Inset)Scatchiard plot for the specific [3H]glibenclamide binding to WGA-purified material. B/F, bound/free. [3HlGlibenclamide was at 1 nM.

Photoaffinity labeling was performed on intact and solubi-lized membranes and on purified receptor preparations.Typical results presented in Fig. 5 indicate that radioactivity

2

200

11692

66 _

45 _

FIG. 4. NaDodSO4/polyacrylamide gel electrophoresis of thepurified [3H]glibenclamide receptor. Electrophoresis was carried outin 4-12% polyacrylamide gels under nonreducing and reducingconditions. Standards were myosin (Mr 200,000), 8-galactosidase(Mr 116,000), phosphorylase b (Mr 97,000), bovine serum albumin(MW 66,000), ovalbumin (M, 45,000), carbonic anhydrase (Mr 31,000),and STI (Mr 21,000). Molecular weights are shown as Mr X 10-3. TheMr 150,000 polypeptide is indicated. Lane 1, active peak from theWGA fraction after a subsequent chromatography through a mixtureof AMP-agarose/GMP-agarose/HA-Ultrogel. Lane 1, treated with2-mercaptoethanol; lane 2, same purified material without 2-mercaptoethanol treatment. Lanes 1 and 2 were silver stained.

A B C D E F G H11 T-.-I. T -

150-

Front -

_~~~~~~~~~~~~~~~~~~~~~~~4_~ :I_.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1

FIG. 5. Autoradiographic pattern ofphotoaffinity labeling carriedout 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 B,P, E, and G), andpresence (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,without 2-mercaptoethanol. The Mr 150,000 polypeptide is indicated(as Mr X 10-3).

was specifically incorporated into a polypeptide of Mr150,000 ± 5000 (Fig. 5). Gel patterns were identical in thepresence and absence of 2-mercaptoethanol.

DISCUSSION

Sulfonylurea receptors are present in relatively largeamounts in insulinoma cells (150 fmol/mg of protein); in thesecells the receptors have clearly been shown to be associatedwith ATP-regulated K+ channels (7). Sulfonylurea receptorsare also present in other cell types, such as cardiac cells (8)or neuronal cells (9, 19, 20). Pig brain cortex membranescontain a large amount of sulfonylurea receptors (=400fmol/mg of protein) and have actually a higher bindingcapacity for this category of drugs than insulinoma cellsthemselves (150 fmol/mg of protein) (7). Structure-functionrelationships in the sulfonylurea series are the same formembranes of insulinoma cells and brain membranes (Fig.2B).

Sulfonylurea-binding sites in brain membranes have notyet been related with functional neuronal ATP-regulated K+channels. However, because the pharmacological propertiesof sulfonylurea-binding sites seem to be the same in neuronalcells as in insulin-secreting cells and cardiac cells in whichsulfonylurea receptors have been associated with ATP-regulated K+ channels, it seems likely that, also in this typeof cell, sulfonylurea receptors will be associated with ATP-regulated K+ channels. The physiological function of theATP-regulated K+ channel in brain cells remains to beelucidated. However, this channel may play an importantrole in ionic disorders associated with ischemia in the brain.The first step following ischemia corresponds to an elevationof the interstitial K+ concentration in the brain, indicatingthat a K+ effiux pathway has been opened (21). This intra-cellular K+ loss may well be a direct consequence of thedecrease ofthe intracellular concentration ofATP linked to theischemic situation. A good candidate as a transport systemresponsible for the K+ loss would then be the ATP-regulatedK+ channel, which is known to be in an open state at lowintracellularATP concentrations. Such a view finds support inthe fact that the initial phase of the K+ leak is slower inischemic animals put in hyperglycemic situations (i.e., pre-sumably with higher levels of intracellular ATP) and acceler-ated in ischemic animals in hypoglycemic situation (21). Otherfunctions could be ascribed to ATP-regulated K+ channels inthe brain, particularly in glucose-sensing hypothalamic neu-rones in charge offeeding regulation (22). These neurones mayemit electrical signals upon glucose perfusion in a way similarto beta cells in the pancreas.

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9820 Neurobiology: Bernardi et al.

This paper reports the extensive purification of brainsulfonylurea-binding sites (2500-fold). The strategy for thispurification has used the idea that (i) this receptor, if it isindeed associated with an ATP-regulated K+ channel, will beretained on an ADP-agarose affinity column since it is known(23-26) that ADP itself regulates the activity of this type ofchannel, (ii) the binding protein, as other binding proteins fortoxins or cardiovascular drugs or diuretics that alter thefunction of other types of channels, is a glycoprotein and willtherefore be retained on a lectin column, (iii) the receptor willnot be retained on AMP-agarose or GMP-agarose columnsbecause these mononucleotides are known to be withouteffect on the activity of the ATP-regulated K+ channel in betacells and cardiac cells (23-25).The solubilized receptor is analogous in its pharmacolog-

ical properties to the membrane receptor. It has a similaraffinity (Kd = 0.8 + 0.3 nM) for glibenclamide and a similarrank order for the recognition of different drugs in thesulfonylurea series.The purified sulfonylurea-binding component appears as a

single band with a Mr of 150,000 ± 10,000. The mobility of thisband is unaltered by reduction. If the stoichiometry is of onesulfonylurea-binding site per binding component of Mr150,000, one can calculate that the maximal binding capacityof the pure receptor should be 6.7 nmol/mg of protein. Ourbest preparations only had a specific activity of 1 nmol/mg ofprotein. Several explanations can be given for the fact thatthe specific activity is not high enough whereas the gelpattern seems to be very satisfactory. The first one is ofcourse that the putative ATP-regulated K+ channel is anoligomeric protein and that the stoichiometry is of less thanone glibenclamide-binding site per channel protein. The mostlikely one is that there is an underestimation of the finalbinding capacity, which is probably due to the relatively shorthalf-life of the purified receptor (12 hr at 40C). Since it takesabout that length of time to purify the sulfonylurea-bindingprotein, then one should never expect an activity better thanabout 3 nmol/mg of protein. The protein with a Mr of 150,000is probably a mixture of active and inactivated binding com-ponents. Previous purifications of voltage-dependent Na'and Ca.2 channels (15-17) have also led to final specificactivities that, for the same reasons, were well under theexpected values.One very convincing aspect of the results is that the

molecular weight of the purified sulfonylurea-binding com-ponent is very similar if not identical to the molecular weightof the protein band that has been identified as the sulfonyl-urea receptor by direct affinity labeling with [3H]gliben-clamide (Fig. 5). The same type of affinity labeling hasrecently been independently tried on insulinoma cells. Thelabeled band identified after gel slicing (instead of autoradi-ography as shown in Fig. 5) has suggested. a Mr of 140,000(27). The similarities of the molecular weights found withbrain- and beta cell-binding components of sulfonylureasprovide another indication that in both cases they correspondto ATP-regulated K+ channels.The subunit constitution of the sulfonylurea receptors

seems to be different from that found for other identified K+channels. A family of voltage-dependent K+ channels hasrecently been cloned by using the Shaker mutation in Dro-sophila. Drosophila K channels of the A type have beenfound to have a Mr of 70,000 (28). Interestingly, the size ofthis channel protein is the same as that found for voltage-sensitive K+ channels identified in mammalian brain with twospecific toxins-i.e., dendrotoxin (29-31) and mast celldegranulating peptide (30, 31).The purification and elucidation of the subunit structure of

the ATP-regulated K+ channel open the way for a molecularanalysis of the ATP-regulated K+ channel at the DNA level.

It will be particularly interesting to see whether this type ofchannel protein, in spite of the differences in size with otherpurified channels, also belongs to the superfamily of channelstructures that already includes voltage-sensitive Na', Ca2+,and K+ channels (32).

We are grateful to Hoechst-Roussel Pharmaceutical Inc. forgenerous gifts of [3H]glibenclamide, glibenclamide, HB699, andtolbutamide and to Boehringer Ingelheim, Laboratoires Roche,Shering-Plough Corp., and Laboratoires Servier for gliquidone,glibornuride, glisoxepide, carbutamide, and gliclazide. We thank Dr.J. Kitabgi for gifts of chlorpropamide and glipizide and Dr. A.Lombet for fruitful discussions. Thanks are due to M. Tomkowiakand C. Roulinat-Bettelheim for expert technical assistance. Thiswork was supported by the Centre National de la RechercheScientifique, the Fondation pour la Recherche Mddicale, and theMutuelle Gdndrale de l'Education Nationale.

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