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Brain Research, 552 (1991) 119-123 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116693J BRES 16693

Purification of a nicotinic acetylcholine receptor from rat brain by affinity chromatography directed at the acetylcholine binding site Andrew J. Dwork and Jeremiah T. Desmond Division of Neuropathology, Columbia University; Department of Neuropathology and Neurotoxicology, New York State Psychiatric Institute, New York, NY 10032 (U.S.A.) (Accepted 8 January 1991)

Key words: Acetylcholine receptor; Affinity chromatography; Affinity purification; Brain; Nicotine; Nicotinic receptor; Rat

We have purified a nicotinic acetylcholine receptor from rat brain by use of an acetylcholine affinity resin commonly employed for the purification of nicotinic acetylcholine receptor from electric tissue. Receptor, specifically eluted with nicotine, bound (-)-[3H]nicotine with a dissociation constant of --21 nM. Binding was inhibited by carbamylcholine but not by a-bungarotoxin. Polyacrylamide gel electrophoresis yielded two protein bands, of apparent mol.wts. 80,400 and 52,400. These results provide independent confirmation of the subunit size and composition reported for rat brain nicotinic receptor isolated by immunoaffinity methods and demonstrate a method of purification that can be performed with commercially available reagents. INTRODUCTION Several m e t h o d s have been e m p l o y e d for the affinity purification of putative nicotinic acetylcholine receptors ( N A C H R ) from brain. Immunoaffinity c h r o m a t o g r a p h y has been used successfully to isolate N A C H R from chick 2t'26, rat 23, bovine 25, and h u m a n 25 brains. The

resin for the purification of N A C H R from rat brain. A similar a p p r o a c h has b e e n r e p o r t e d recently by a n o t h e r l a b o r a t o r y 13, but the p r o c e d u r e and results differ considerably from ours. MATERIALS AND METHODS

principal limitations of this technique are that it requires a supply of m o n o c l o n a l a n t i b o d y with affinity for the r e c e p t o r to be purified, and that harsh conditions are r e q u i r e d for elution of the receptor. Affinity columns e m p l o y i n g a - b u n g a r o t o x i n have been used to isolate proteins from rat 1t'23, mouse is, chicken 5't4'16, and gold-

Sprague-Dawley rat brains, frozen and shipped in liquid nitrogen, were from Zivic-Miller (Zelienople, PA). Affi-Gel 401 was from Bio-Rad (Richmond, CA). Acrylamide, bis-acrylamide, and sodium dodecyl sulfate were from BDH (Poole, England). (-)[N-methyl-3H]nicotine was from Amersham (Arlington Heights, IL). Hydrofluor scintillation fluid was from National Diagnostic (Manville, NJ). Other reagents were from Sigma (St. Louis, MO).

fish 9 brains, but the bungarotoxin-binding proteins so o b t a i n e d are clearly distinct from receptors with high affinity for acetylcholine and nicotine 9't6"23. A n affinity column employing a nicotine analogue has been used to isolate a single-subunit p r o t e i n from rat brain 1, but further work on this p r o t e i n has not been reported. N A C H R from electroplax is purified by affinity chrom a t o g r a p h y either with immobilized curarimimetic toxin (reviewed in ref. 10) or with immobilized acetylcholine 15. A l t h o u g h two reports mention unsuccessful attempts to use the latter resin for purification of N A C H R from chick 16 and goldfish 9 brain, this was not the main focus of these papers. We r e p o r t here the successful use of this

Assay for binding of (-)-[3H]nicotine All solutions and samples are kept on ice. Detergent-solubilized receptor is diluted into 3-[N-morpholino]propanesulfonic acid (MOPS) buffer, pH 7.4, with NaCI, ethylenediaminetetraacetic acid (EDTA), and Triton X-100 to give final concentrations of 45-50 mM MOPS, 100 mM NaCI, 1 mM EDTA, 1% Triton X-100, and no more than 1.7 mg protein per ml. One hundred/A aliquots of this dilution are placed into 5 ml polypropylene tubes. To each tube is added 50 pl of buffer (50 mM MOPS, 100 mM NaC1, 1 mM EDTA, pH 7.4) with or without 500 pM unlabeled (-)-nicotine or carbamylcholine. After 30 min, 100/A of (-)-[3H]nicotine (60-72 Ci/mmol) in the same buffer is added to each tube. After 60 min, the samples are diluted with 4 ml dilution buffer (10 mM MOPS, 1 mM EDTA, 0.2% Triton X-100, pH 7.4) and immediately filtered under vacuum through Whatman GF/B filters, presoaked overnight in 0.5% polyethylenimine. The filters are then washed 3 times with 4 ml dilution buffer, and scintillation counting is performed in Hydro-

Correspondence: A.J. Dwork, Department of Neuropathology and Neurotoxicology, New York State Psychiatric Institute, 722 West 168th St., Box 62, New York, NY 10032, U.S.A.

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fluor. Specific binding is taken as the difference between that obtained in the presence and absence of unlabeled (-)-nicotine or carbamylcholine, which at 100]~M final concentration yield identical results.

20 18

16

Affinity purification The entire procedure is performed at 4 °C, and all solutions contain 0.5 mM phenylmethylsulfonyl fluoride. In a Waring blendor, 160 g of partially thawed rat brains (previously stored in liquid nitrogen) are homogenized in 640 ml of 1 mM EDTA, pH 7.4, for 10 s on 'low'. The homogenate is centrifuged at 700 g for 10 rain. The supernatant is set aside, and the pellet is resuspended in 480 ml EDTA and centrifuged as above. The supernatants are pooled and centrifuged for 200 min at 16,000 g. The resulting pellet ( - 9 5 ml) is combined with 10 mM sodium phosphate, 2 M NaCI, 1 mM EDTA, pH 7.0, to a final volume of 175 ml. This is homogenized in a Brinkmann Polytron (setting '5') for 45 s, and Triton X-100 is added to a final concentration of 0.1%. The suspension is stirred for 1 h and then centrifuged for 30 min at 120,000 g. The pellet is homogenized as above with 50 mM MOPS, 1 mM EDTA, 100 mM NaCI, 0.02% NaN 3, pH 7.4, in a final volume of I(X) ml. Triton X-100 is added to a final concentration of 3%, and the homogenate is stirred overnight and then centrifuged at 120,000 g for 90 min. The clear supernatant (50-80 ml) is carefully removed and dialyzed for 3 h against 20 mM MOPS, 40 mM NaCI, 1 mM EDTA, 0.02% NaN 3, 1% Triton X-100, pH 7.4. Affinity gel (approx. 5 ml) is prepared fresh and assayed exactly as described '5. except that bromoacetylcholine (synthesized and assayed as described in ref. 6) is used at 20 mM. Invariably, all Affi-Gel sulfhydryl groups react with bromoacetylcholine. Atropine is added to the dialyzed extract to a final concentration of 10/tM. Affinity gel is added in a 1:20 ratio to extract, and the mixture is stirred for - 2 2 h. The affinity gel is then recovered by centrifugation and washed 3 times with 10 mM MOPS, 40 mM NaCI, 1 mM EDTA, 1.0% Triton X-100, 10 I~M atropine, pH 7.4, by shaking followed by centrifugation. The gel is then resuspended in the same buffer and transferred to a column (1 cm i.d.). The column is then washed for 18-20 h with the same solution, except with 100 mM NaCI and 1.5% Triton X-100, at a flow rate of - 0 . 2 ml/min, allowing collection of 200-250 ml of buffer. Specific elution is then performed with 10 mM (-)-nicotine in 10 mM MOPS, 90 mM NaCI, 1 mM EDTA, 1.5% Triton X-100, pH 7.4 (no atropine), at the same flow rate, and at least 5 fractions of 2 ml are collected. Portions to be used for binding assay are dialyzed extensively (> 24 h) to remove nicotine. Unused aliquots are stored in liquid nitrogen.

RESULTS

Only - 1 5 % of initial (-)-[3H]nicotine binding sites remained in solution after overnight incubation with the affinity resin. Exhaustive dialysis (mol.wt. cutoff = 25,000) yielded no additional binding sites, indicating that the loss of binding sites was not due to the presence of soluble ligand leached from the column. If a 'dummy' gel, prepared by reacting Affi-Gel 401 with iodoacetamide, is used in place of the affinity gel, there is no detectable loss of binding sites. Therefore, the loss of binding sites from solution is due to specific binding to the affinity resin. Approximately 1% of applied nicotinic sites were contained in the early wash fractions; later wash fractions contained no nicotine-binding activity. Binding sites eluted sharply with the addition of nicotine. Total yield, based on specific binding of 10 nM (-)[3H]nicotine and ignoring the apparent change in KD (see

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I I 120

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Fig. 1. Top: specific binding of (-)-[~H]nicotine to 100~1 aliquots of 1:16 dilution of specifically eluted NACHR, assayed as described in Materials and Methods. Each symbol represents mean + S.E.M. for quadruplicate determinations. Bottom: Scatchard plot of same data.

below), was 9-20% of bound sites. Most of this was in the second and third nicotine fractions. A small additional amount of binding activity ( - 5 % of the total recovered) could be obtained after the initial elution by allowing the column to equilibrate for 1 h with 10 mM (-)-nicotine. Scatchard analysis (Fig. 1) of the specific binding of (-)-[3H]nicotine to the purified product yielded a linear plot and a dissociation constant of 21 + 2 nM (mean +_ S.D. for 3 determinations on different preparations). The most concentrated fractions contained 2.9-5.4 nM (average = 3.5 nM) nicotine-binding sites; protein concentration was below the limits of detection by Lowry assay (~0.01 mg/ml). Binding of (-)-[3H]nicotine to purified receptor was --t00% specific at all concentrations and was inhibited equally by 100 /~M nicotine or 100 /~M carbamylcholine. Binding of 10 nM (-)-[3H]nicotine was 48% inhibited by 1 ,uM carbamylcholine and was not at all inhibited by 10/*M a-bungarotoxin. Scatchard analysis of binding of (-)-[3H]nicotine to the detergent extract, prior to affinity purification, yielded a linear plot with a dissociation constant of 11 +- 2 nM and

121

A

B

C

97.4

45

Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis tz of samples containing: A: 4.5/~1 of specifically eluted NACHR (24 fmol binding sites); B: sample buffer alone; C: 4.5/~1 of last wash fraction before specific elution. Silver stain. Positions of mol.wt. (in kDa) standards are indicated.

a Bin,ix of 105 + 3 fmol/mg protein (mean + S.D. for 3 determinations on different preparations). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis 12 (Figs. 2, 3) showed two bands with apparent mol.wts. 80,400 + 900 and 52,400 + 900 (mean + S.D. of 15 determinations on 8 preparations), only in the specifically eluted fractions. Much fainter contaminating bands were often present, but unlike the major bands, these were also present in the final wash fraction, which lacked any nicotine-binding activity. The 80,400 and 5 2 , 4 0 0 M r proteins were thus specifically eluted by nicotine and specifically associated with nicotine-binding activity.

A

B

Fig. 3. Electrophoresis as in Fig. 2, same preparation. After elution of receptor and complete washout of nicotine at 4 °C, affinity column was washed at room temperature with 4% sodium dodecyl sulfate. A: 4.5 pl of same NACHR fraction as in Fig. 2. B: 4.5 pl of sodium dodecyl sulfate wash.

DISCUSSION

We have demonstrated that a N A C H R from rat brain, obtained by affinity chromatography directed at the acetyicholine binding site, has high affinity for agonist and little or none for a-bungarotoxin. It is composed of two subunits with apparent moi.wts, of approx. 80,400 and 52,400. This result is virtually identical with that obtained for receptor purified by immunoaffinity methods. Using an immobilized monoclonal antibody, Whiting and Lindstrom 23 obtained from rat brain a NACHR composed of subunits with apparent mol.wts. 79,700 and 51,700. It is therefore likely that we have obtained the same receptor. This subunit composition is also supported by the finding that the N-terminal sequences of the heavy 2° and light ~7 subunits obtained by immunoaffinity purification are consistent with their being encoded by two cloned genes, respectively a48 and f127. The proteins encoded by these genes form functional NACHR when expressed together in Xenopus oocytes3. NACHR from bovine brain 25 and two subtypes of NACHR from chicken brain 26 are each composed of two types of subunit. In all cases, nicotinic affinity labels bind to the larger subunit 24'25. Our results differ from those of Nakayama et al. 13. Using membranes solubilized in Lubrol PX, these investigators performed successive chromatography on DE-52, Affi-Gel-acetylcholine, and hydroxylapatite. They obtained 4 major protein bands, of M r 53,000, 67,000, 80,000, and 108,000. The explanation for their additional bands is uncertain. Since yields in all purifications are quite low (4.7% by Whiting and Lindstrom23; 15% by Nakayama et a1.13; 9-20% by us), it is possible that different subtypes of N A C H R are being purified by different procedures. Two different forms of NACHR have been identified in chick brain 26 and numerous candidate genes for both acetylcholine-binding and 'structural' subunits of rat brain nicotinic receptor have been cloned and their transcription in rat brain demonstrated by in situ hybridization (see ref. 4). On the other hand, the protein bands obtained by Nakayama et al. ~3 may include contaminants. We routinely wash the affinity column with - 8 0 column vols. of buffer and elute essentially all of the receptor in - 3 column vols. of buffer with nicotine. Even so, several more faintly stained bands are present, but these are clearly contaminants since they are seen also in the fraction collected immediately prior to elution by nicotine, which contains no nicotine-binding activity (Fig. 2). We had initially washed with only 10-20 column vols. (comparable to Nakayama et al.~3). Elution of receptor was similar to that obtained after extensive washing, but polyacrylamide gel electrophoresis revealed many other

122 bands, of c o m p a r a b l e intensity to the specifically eluted ones, both in the nicotine-eluted fractions and in the previous wash fraction. The potential for contamination is further u n d e r s c o r e d by the finding that after washing the gel and eluting the r e c e p t o r according to our present protocol, washing the gel with 4% sodium dodecyl sulfate yields at least 25 strongly staining bands, closely spaced along the entire length of the gel (Fig. 3). The K D of 21 n M that we o b s e r v e d for the binding of (-)-[3H]nicotine to affinity-purified r e c e p t o r is approximately double the K o of 11 nM for the Triton extract prior to the affinity purification step. T h e r e are several possible explanations for this difference. The a p p a r e n t K D could be affected by some c o m p o n e n t of the extract which is subsequently removed. Alternatively, during the affinity purification or the subsequent dialysis to remove nicotine, the r e c e p t o r could b e c o m e slightly d e n a t u r e d , resulting in the o b s e r v e d lowering of affinity for nicotine. Finally, it is possible that the Triton extract contains a mixture of proteins with slightly differing affinities, sufficiently close to yield an a p p a r e n t l y linear Scatchard plot, and that one or m o r e of these are lost during the r e m a i n d e r of the procedure. O u r dissociation constants are higher than the K D of 1.5 nM for binding of (+)-[3H]nicotine to immobilized r e c e p t o r o b t a i n e d by Whiting and Lindstrom 22, but c o m p a r a b l e to values r e p o r t e d for binding of ( - ) [3H]nicotine to rat brain m e m b r a n e s (0.2-20 nM, re-

viewed in ref. 27) and to that for the binding of (+)-[3H]nicotine to rat brain m e m b r a n e s solubilized in 1% Triton X-100 (11 nM, c o m p a r e d to a value of 5.6 nM r e p o r t e d in the same p a p e r 2 for intact m e m b r a n e s ) . The 48% inhibition that we o b s e r v e d of 10 nM (-)[3H]nicotine binding to purified r e c e p t o r by 1 ~ M carbamylcholine is c o m p a r a b l e to the IC50 of 0.95/~M o b t a i n e d by Whiting and Lindstrom 22 for inhibition by this c o m p o u n d of binding of 10 nM (+)-[3H]nicotine to immobilized N A C H R from rat brain. The concentration of protein in our specifically eluted fractions is below - 0 . 0 1 mg/ml, the lower limit of our Lowry assay. The most c o n c e n t r a t e d fractions thus represent at least a 3500-fold purification over the Triton extract. We estimate from the a p p e a r a n c e of the gels that the specifically eluted bands account for at least 50% of total protein, which, assuming a mol.wt, of - 1 5 0 , 0 0 0 per binding site 19, would represent - 3 0 , 0 0 0 - f o l d purification. The purification is fairly simple, requires only one column, and is p e r f o r m e d with commercially available materials. The p r o c e d u r e should be readily applicable to the purification of N A C H R from the brains of other species.

REFERENCES

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1 Abood, L.G., Latham, W. and Grassi, S., Isolation of a nicotine binding site from rat brain by affinity chromatography, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 3536--3539. 2 Abood, L.G., Reynolds, D.T. and Bidlack, J.M., Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature, Life Sci., 27 (1980) 1307-1314. 3 Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S. and Patrick, J., Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7763-7767. 4 Boulter, J., O'Shea-Greenfield, A., Duvoisin, R.M., Connolly, J.G., Wada, E., Jensen, A., Gardner, P.D., Ballivet, M., Deneris, E.S., McKinnon, D., Heinemann S. and Patrick, J., a3, a5, and f14: Three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster, J. Biol. Chem., 265 (1990) 4472-4482. 5 Conti-Tronconi, B.M., Dunn, S.M.J., Barnard, E.A., Dolly, J.O., Lai, EA., Ray, N. and Raftery, M.A., Brain and muscle nicotinic acetylcholine receptors are different but homologous proteins, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 5208-5212. 6 Damle, V.N., McLaughlin, M. and Karlin, A., Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica, Biochem. Biophys. Res. Commun., 84 (1978) 845-851. 7 Deneris, E.S., Connolly, J., Boulter, J., Wada, E., Wada, K., Swanson, L.W., Patrick, J. and Heinemann, S., Primary structure and expression of f12: a novel subunit of neuronal nicotinic acetylcholine receptors, Neuron, 1 (1988) 45-54.

Acknowledgements. This work was supported by a grant from the Smokeless Tobacco Research Council and by NIH Grant BRSG-07RR05395. We are grateful for the assistance of Karin A. Malkin, who was supported by a Student Fellowship from the Parkinson's Disease Foundation.

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and Lindstrom, J., cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor, Neuron, 1 (1988) 241-248. Seto A., Arimatsu Y. and Amano T., Subunit structure of a-bungarotoxin binding component in mouse brain, J. Neurochem., 37 (1981) 210-216. Whiting P., Cooper, J., Conroy, W.G. and Lindstrom, J., Subunit stoichiometry of neuronal nicotinic acetylcholine receptors, Soc. Neurosci. Abstr., 15 (1989) 496. Whiting, P., Esch, F., Shimasaki, S. and Lindstrom, J., Neuronal nicotinic acetylcholine receptor fl-subunit is coded for by the cDNA clone a4, FEBS Lea., 219 (1987) 459-463. Whiting, P.J. and Lindstrom, J.M., Purification and characterization of a nicotinic acetylcholine receptor from chick brain, Biochemistry, 25 (1986) 2082-2093. Whiting, P. and Lindstrom, J., Pharmacological properties of immuno-isolated neuronal nicotinic receptors, J. Neurosci., 6

(1986) 3061-3069. 23 Whiting, P. and Lindstrom, J., Purification and characterization of a nicotinic acetylcholine receptor from rat brain, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 595-599. 24 Whiting, P. and Lindstrom, J., Affinity labelling of neuronal acetylcholine receptors localizes acetylcholine-binding sites to their fl-subunits, FEBS Lett., 213 (1987) 55-60. 25 Whiting, P.J. and Lindstrom, J.M., Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monocional antibodies, J. Neurosci., 8 (1988) 3395-3404. 26 Whiting, P.J., Liu, R., Morley, B.J. and Lindstrom, J.M., Structurally different neuronal nicotinic acetylcholine receptor subtypes purified and characterized using monoclonal antibodies, J. Neurosci., 7 (1987) 4005-4016. 27 Wonnacott, S., Brain nicotine binding sites, Human Toxicol., 6 (1987) 343-353.