Solubilization of the Muscarinic Acetylcholine Receptor by I Sodium Cholate: Stabilization of the Receptor by Muscarinic Hgands
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\ i Biomedical Research 3 (6) 695-698, 1982 Solubilization of the muscarinic acetylcholine receptor by I sodium cholate: Stabilization of the receptor by muscarinic Hgands TATSUYA HAGA, TOSHIHIDE NUKADA and KAZUKO HAGA Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan ABSTRACT Forty to fifty percent of the muscarinic receptor in membrane preparations of porcine caudate nucleus was solubilized by sodium cholate in its active form when the mem- branes were pretreated with carbamylcholine or other muscarinic ligands. The binding activity of solubilized receptors was assayed by using [3H]quinuclidinylben- zylate ([3H]QNB) after removal of cholate and carbamylcholine. Without the pre- treatment, sodium cholate abolished most of the binding activity. The solubilized receptor had a high afiinity for [3H]QNB with a dissociation constant of 58 pM and an affinity for muscarinic ligands similar to that of the membrane receptor. The muscarinic receptor has been studied in —80°C until use. In the standard procedure of detail with respect to its binding with muscarinic solubilization, the microsomal preparation ligands by using [3H]-labeled ligands (3, 12) and (about 5 mg protein per ml) was incubated with several subclasses with different affinities for l0 mM carbamylcholine in 50 mM Tris-HCI agonists (2) or selective antagonists (8) have buffer (pH 7.5) at 30°C for 10 min, then a half been reported. It is not known, however, volume of 3 ‘X, sodium cholate in the Tris buffer whether these subclasses represent several types was added and the incubation continued for 5 of receptors which differ as chemical entities, or min at 30°C. The suspension was centrifuged several states of a single receptor which differ in for 1h at l65,000g and the supernatant was conformation and/or in interaction with other passed through Sephadex G-50 columns which components in the membranes. The direct were pre-equilibrated with the Tris buffer. An answer to this question would be obtained aliquot of the void volume fraction was incubated by purification and reconstitution of the with 2 to 2.5 nM [3H]QNB in the Tris buffer at receptor. The purification of the receptor 30°C for 30 min and the bound form of [3H]QNB should be preceded by solubilization of the was separated by using small columns of receptor from membranes. Two kinds of Sephadex G-50 as described previously (5, 6). detergents, digitonin (for review, see l0) and The binding of membrane receptor with PH]- lysophosphatidylcholine (5) have been reported QNB was assayed by using glass fiber filter paper to partly solubilize the receptor but common (Whatman GP/C). Protein concentrations were detergents like Triton X 100 or sodium cholate determined by the Lowry method using bovine have failed to solubilize it in its active form (9, serum albumin as a standard. ll). This paper is concerned with a finding Fig. l summarizes the effects of pretreatment that the muscarinic receptor can be solubilized of membranes with muscarinic ligands and other in its active form when membranes are pre- agents on solubilization of the muscarinic treated with muscarinic ligands and then treated receptor by sodium cholate. About 40% of with sodium cholate in their presence. [3H]QNB binding activity in the membranes Microsomal fractions prepared from porcine was recovered in the supernatant when the caudate nucleus by the usual method were membranes were pretreated with carbamylcho- suspended in 0.32M sucrose and stored at line and solubilized by sodium cholate in the 696 'RIhMh&TZNUKADAandKsHAGA °H-QNB Binding of Solubilized Fraction (fmol) 0 20 40 60 80 Additives in the i T 0'" __ T.’ __ 77;‘ "' ”""'”""'iilZ ---1 ll Extraction Medium ii 1M NaCl; 1 miVi GTP . J if W 1 mM NEM 1 m|V! Carbachol i +1 M NaCI 7 i _ + 1 mivi GTP — JL_ +1mMNEM-A I 0 1 ;iM Atropine - 0 0.1mm Pridinol 9 I T F ' °H-QNB Binding of original membranes : 151 fmol L __ I ____, *- -4 , -+-- _. - ~ L .... _. 2 Fig. l Effects of the pretreatment of membranes with muscarinic ligands and other reagents on the solubilization of the muscarinic receptor by sodium cholate. Membrane preparations were first in- cubated at 30°C for 10 min in Tris-HCl buffer (pH 7.5) with the reagents shown in the figure. The concentrations of the reagents in the prein- cubation mixture were 1.5 times higher than those shown in the figure. To the pretreated membranes was added a half volume of 3% sodium cholate. The suspension was incubated for 30 min at 0°C and centri- fuged at l65,000g for 60 min. The supernatant was passed through a Sephadex G-50 column and the void volume fraction was used for the [3H]QNB binding assay as described in the text. The specific binding per 0.072 mg of original membrane protein was determined in the presence and absence of 1 /1M atropine and plotted in the figure. presence of carbamylcholine. On the other of l M NaCl without addition of carbamylcho- hand, less than 10% of the binding activity was line. Coexistence of NaCl and carbamylcholine, recovered in the supernatant when the mem- however, did not increase the yield any more. branes were solubilized in the absence of car- It is possible, but remains to be proven, that Na bamylcholine. In this and the following stabilizes the receptor by binding to the same experiments, the supernatant was passed through site as muscarinic ligands. a Sephadex G-50 column to remove compounds GTP did not affect the recovery of [3H]QNB of small molecular weights including carbamyl- binding sites with or without carbamylcholine. choline and cholate, and then the void volume The treatment of membranes with N-ethyl- fraction was used for the binding assay. About maleimide caused a decrease of the [3H]QNB 70% of the binding activity in the void volume binding activity in the cholate extracts to 30 to fraction was recovered in the supernatant after 50 % of the control, although the same treatment centrifugation at l65,000g for 1 h. did not affect the [3H]QNB binding activity of The effect of carbamylcholine was obtained the membrane receptor but enhanced its affinity also with atropine or pridinol indicating that the for agonists (1). On the other hand, treatment stabilization of the muscarinic receptor was with N-ethylmaleimide resulted in an almost attained by muscarinic ligands irrespective of complete loss of [3H]QNB binding activity in whether they were agonists or antagonists. the preparation solubilized by lysophosphatidyl- About 30% of [3H]QNB binding activity was choline (7). recovered in the supernatant when the mem- Fig. 2 shows the effect of cholate concentra- branes were solubilized by cholate in the presence tions on the solubilization of [3H]QNB binding SOLUBILIZATION OF MUSCARINIC RECEPTOR 697 1-1' W - i i ' 17*? P—— i’ i i * i * r—~ wt m F Ya- 1.5 .* ~‘l.5 ii‘ 1i i; 13 eptor 11 C) .,.4 1000 ‘T 12 B (.0 ni- Ii 9 S ': l O )(0,0) 5_ Recb‘zed 7 i O3 1,, 6 -T /mpmo . 5 T j Azao(A) ‘F1?‘ un- (nd'ngQ9‘ .601 . O123 2 \ Choforuate-So z‘U 5- T Q. 4; ,4 J, 3H-QNBB 4 10 M atropine 5 5 is T7 0 an 9 0100011 0 -,9 I ........_ 9 50 pKd for Membrane Receptor 0 i 2 3 4 Cholate (%) Fig. 3 Comparison of affinity for muscarinic ligands between membrane-bound and cholate- Fig. 2 Effects of sodium cholate concentrations on solubilized receptors. The value of pKd (: —-log Kd) the solubilization of the muscarinic receptor. Mem- was calculated from the following equation: Kd= branes were treated with l0 mM carbamylcholine for IC5n/(1+([3H]QNB)/Kdi‘), where IC5n is the con- 10 min at 30°C, and the subsequent solubilization centration of the ligand giving half maximal inhibi- and [3H]QNB binding assay were carried out as tion of [3H]QNB binding and Kdi‘ is the dissociation described in the text except that cholate concentra- constant of the binding of the receptors with [3H]- tion was varied as indicated. Absorbance was QNB (10 pM for the membrane receptor and 58 pM measured of the sample eluted in the void volume for the receptor solubilized by cholate). ([3H]QNB) fraction of a Sephadex G-50 column. In the case of is the concentration of [3H]QNB used (0.1 nM for 100% recovery, the [3H]QNB binding activity and the experiment with the membrane receptor and 2 to protein concentration in the void volume fraction 2.5 nM for the experiment with the solubillized should be 3.8 pmol/ml and 1.8 mg/ml, respectively. receptor). The correlation equation was Y: 0.42+0.88X with a correlation coefificient of 0.99. The ligands used were as follows: 1, carbamylcholine; sites. The recovery of [3H]QNB binding 2, acetylcholine; 3, arecoline; 4, cz's-2-methyl-di- activity was maximal at the cholate concentra- oxolane; 5, oxotremoline; 6, diphenidol; 7, prometa- tion of 1%. Further increase of the cholate zine; 8, methixene; 9, triphenidyl; 10, atropine; 11, concentration was accompanied by a decrease in scopolamine; 12, isopropamide; 13, [3H]QNB the recovery of the binding activity although the amount of solubilized protein was increased. The recovery of [3H]QNB binding sites at 1% [3H]QNB binding and compared with those of cholate was 46i13 % (average i SD; n=9) of the membrane receptor (Fig. 3). The Kg values that in membrane preparations. The recovery obtained for the solubilized site correlated well varied from 20 to 60% but usually ranged (r=0.99) with those obtained for the membrane between 40 and 50%.