PART
Iv.
MECHANISM ACROSS SYNAPTICMEMBRANES
PERMEATION
PURIFICATION OF ACETYLCHOLINE RECEPTOR FROM TORPEDO CALIFORNICA AND ITS INCORPORATION INTO PHOSPHOLIPID VESICLES" Mark G. McNamee, Cheryl L. Weill, and Arthur Karlin Department of Neurology College of Physicians and Surgeons Columbia University N e w York, New York 10032
The acetylcholine receptors (AChR) translate the binding of acetylcholine into increases in ion permeabilities of synaptic membranes. The receptor protein can be solubilized from the membrane by nonionic detergents and purified by affinity chromatography. The isolated protein retains the specific binding properties attributed to the AChR in situ and is being used to deduce the molecular properties of the AChR. Purification and characterization of the AChR has been the subject of several recent reviews.'-3 A complete molecular description of receptor function must include its interactions with other membrane components. Considerable effort is now being dedicated to the reconstitution of cation permeability control in rnem branes containing AChR'-a and our initial efforts in this area are discussed.
PURIFICATION OF
ACETYLCHOLINE RECEPTOR
Tissue from the electric organ of Torpedo californica is homogenized in 1 mM EDTA (pH 7.4), and the pellet obtained after centrifugation is homogenized in 1 M NaCI-2 m M NaPOa-1 mM EDTA (pH 7.4), sedimented, and resuspended in 1 mM EDTA (pH 7.4), and resedimented. This crude membrane pellet is then extracted for 1 hr at 40 with 3% Triton X-100-50 mM NaCI-10 mM NaPO+-l mM EDTA-3 mM NaN3 (pH 8.0). The supernatant after centrifugation contains on the average 16% of the initial protein, 18% of the acetylcholinesterase ( AChE) , and nearly all of the AChR. The procedure used is identical to that used for extraction of AChR from Electrophorus electricus and complete details may be found elsewhere." The specific activity of the AChR in the detergent extract is approximately 300 pmol of sites per mg of protein, approximately ten times higher than that obtained with Electrophorus. Solubilized AChR is purified by affinity chromatography using p-carboxyphenyltrimethylammonium ion, coupled to Affi-Gel 401 (Bio-Rad Lab., Rich-
* This work was supported by Research Grants NS 07065 from the National Institute of Neurological Diseases and Stroke and BMS75-03026 from the National Science Foundation and by a grant-in-aid from the New York Heart Association, Incorporated. 175
176
Annals New York Academy of Sciences TABLE1 AFFINITY CHROMATOGRAPHIC PURIFICATION F R O M Torpedo californica
OF
RECEPTOR ~~~~~
Protein * Elution Sequence Extracts Input Not bound TNP5Oq TNPl 00 10 mM CARBII in TNP 90 Total recovery
mg 1133 900 6.9 8.5 26.3 942
AChEt
Receptor$
%
nmol
%
nmol
%
100 79.4 0.6
19.2 16.3 0.39 0.21 1.27 18.2
100 85 .O 2.0 1.1 6.6 95
371 0 0 1.5 73.5 75
100 0 0 0.6 19.8 20
0.8
2.3 83
*Protein was determined by the Lowry method with bovine serum albumin as the standard." t Acetylcholinesterase activity was measured by Ellman's p r o c e d ~ r e and ' ~ is expressed as nmoles of catalytic sites assuming 80,000 daltons per catalytic site and 10 mol acetylthiocholine hydrolyzed per min per g enzyme. $ Receptor activity was determined by specific [ 3H]MBTA labeling (see text). 8 Extract (186 ml) from 600 g of tissue in 3% Triton X-100-50 mM NaCl-10 mM NaP0,1 mM EDTA-3 mM NaN, (pH 7.0). 9 TNPxx contains 0.2% Triton X-100,xx mM NaCl, 10 mM NaPO,, 1 mM EDTA, 3 mM NaN, (pH 7.0). 11 Carbamylcholine was removed from fractions by dialysis before assay for AChE and receptor.
mond, Calif.), as the adsorbing ligand and carbamylcholine (CARB) as the eluting ligand. TABLE 1 provides a summary of a typical elution pattern from the affinity column. The AChR can be further purified about 30% by centrifugation in a 5-20% sucrose density gradient containing 0.2% Triton X-100.Typically, the AChR appears as two peaks (FIGURE 1 ) . The faster-moving peak moves as if it were a dimer of the slower peak. The specific activity of AChR is constant across both peaks at about 4 nmol of MBTA sites per mg of protein (see below). Both peaks are resolved from remaining traces of AChE and less than .001% of the pooled AChR fractions is active AChE. CHARACTERlZATION
OF
ACHR
Afinity Alkylation
The AChR can be assayed at all stages of purification by affinity alkylation of dithiothreitol-reduced receptor with 4-(N-maleimido)benzyltri-["H]-methy1ammonium iodide (['HIMBTA), using a modification of the "quick assay" procedure.' The portion of MBTA labeling that is blocked by the principal a-neurotoxin of Naja naja siamensis' is taken to be specific for AChR; all of the ['HIMBTA labeling of purified AChR is blocked by toxin.
McNamee et al. :
0
4
Purification of Acetylcholine Receptor
8
12
16
20
177
24
FRACTION
FIGURE 1. Sucrose density gradient centrifugation of Torpedo AChR. Acetylcholine receptor, purified by affinity chromatography ( 1 mg in 1 ml of TNPSO [see footnote (Q)to TABLE11, 3 nmol MBTA sites/mg) was applied to the top of a 16-1111 5-20% sucrose gradient in TNPlSO and centrifuged at 27,000 rprn for 16.5 hr at 5" in the SW27 rotor of an L2-65B Beckrnan ultracentrifuge. Fractions of 0.5 rnl were collected with an ISCO Model 183 Fractionator and analyzed for percentage of sucrose (-a+), protein (m, 10 pg/unit),I5 AChR ( 0 , 50 nmol MBTA sitedunit), and AChE (A,50 pmol/unit).a Approximately 90% of the protein, AChR, and AChE were recovered. The specific activity of the peak AChR fraction was 4.2 nmol MBTA siteslmg. Toxin Binding A ['Hlmethyl derivative of N . n. siamensis &-toxin was prepared by reductive alkylation of native toxin with formaldehyde and tritiated sodium borohydride,". lo The toxin is labeled to the extent of about 0.5 mol of methyl groups per mol of toxin, and yields about 2000 cpm per pmol under typical counting conditions. The biological activity of [3H]-toxin is indistinguishable from unlabeled toxin by a bioassay using micell and all of the ['HI-toxin is bound by an excess of receptor. The binding of ['HI-toxin to receptor is determined by incubating toxin and AChR for one hour at 2 5 O in 0.2% Triton X-100-100mM NaC1-10 mM NaP04-I mM EDTA-3 mM NaN, (pH 7.0) and then separating toxin-AChR complex from free toxin on Bio-Gel P-30. The bound radioactivity and the protein of the isolated complex are determined. We have found that the specific activity estimated by toxin binding is approximately twice that estimated by [3H]MBTA labeling. For example, a preparation of AChR containing 4.2 nmol of sites susceptible to ['HIMBTA labeling per mg of protein was found to bind 8.4 nmol of 3H-toxin per mg. The 2: 1 ratio does not appear to be dependent on the degree of purification. Since toxin blocks all of ["HIMBTA labeling of purified AChR, there must
178
Annals New York Academy of Sciences
be at least one interacting site of toxin and ['HIMBTA labeling. It has not yet been determined whether or not the additional toxin binding represents a second distinct binding site.
Subunit Structure AChR from Torpedo electric tissue purified by affinity chromatography displays two protein components by polyacrylamide gel electrophoresis under nondissociating conditions in the presence of Triton X-100 (FIGURE2A).
FIGURE2. Polyacrylamide gel electrophoresis of purified AChR. (A, C, and E) Triton X-100 Gels: To samples of AChR from the affinity column (A) and from a subsequent sucrose density gradient centrifugation, second peak (heavier) C and first peak (lighter) D was added solid sucrose to 10% and brornophenol blue. Samples containing 10-12 pg of proteins were applied to 3.6% gels, and run in 0.2% Triton X-100-100 mM Tris-acetate, pH 8 at 3 ma/gel. Note that gel A was not run as long as gels C and E (ink line marks front). (B, D, and F) Sodium dodecyl sulfate (SDS) gels: Samples of AChR obtained as above were dialyzed against 2% SDS-10 mM Tris acetate-2 mM EDTA-pH 8, precipitated, and washed with dry acetone to remove Triton X-100 and dissolved in an appropriate volume of dialysis buffer containing 20 mM DTT for 0.5-1 hr at SO" C. Samples containing 12-18 pg of protein were applied to 5.6% gels and run in 1% SDS-100 mM Tris-acetate, PI-I 8 at 8 ma/geI. Under dissociating conditions, saturated with sodium dodecyl sulfate (SDS) and reduced, this same material gives rise to four polypeptide bands corresponding to apparent molecular weights of 39,000, 48,000, 58,000, and 64,000 daltons (FIGURE 2B) .Iz Sucrose density gradient centrifugation results in the separation of two symmetrical peaks of protein and AChR activity, as described above. The protein from each peak yields a single band on Triton X-100 polyacryl2C and E ) . The relative mobilities of the single bands amide gels (FIGURE in C and D correspond to the relative mobilities of the bands in A. As expected, the dimer has a smaller electrophoretic mobility. The SDS gel pattern of the protein in each peak from the sucrose density gradient centrifugation (FIGURE2D and F) is identical to that obtained with AChR before sucrose density gradient centrifugation [FIGURE 2B). By SDS-gel electrophoresis, it has been shown that the affinity alkylation by 13H]MBTA is directed specifically and exclusively to the subunit of 39,000 daltons.'* This subunit thus bears all or part of the acetylcholine binding site, as previously demonstrated for the subunit of 40,000 daltons in AChR from Electrophorus.' The functional significance of the other components has yet to be ascertained. The specific activity of the most highly purified AChR preparations from
McNamee et al. : Purification of Acetylcholine Receptor
179
Torpedo (about 4 nmol MBTA sites per mg protein) corresponds to a minimal molecular weight of 250,000 daltons per MBTA binding site. The combined apparent molecular weights of the four putative subunits is 209,000.
INCORPORATION
OF
ACHR
INTO
PHOSPHOLIPID VESICLES
Reconstitution of AChR function in model membrane systems is a principal objective of current receptor research. The mechanism by which the binding of acetylcholine and its congeners to the AChR is transduced into a change in membrane cation permeability is not known, and reconstituted vesicles offer the most attractive system for investigating permeability control at the molecular level. We incorporate purified AChR into phospholipid vesicles using a dialysis procedure similar to that used by Racker"' for a variety of membrane systems. The vesicles are formed by dialyzing a solution of AChR and lipids in sodium cholate against detergent-free buffer, under N2, during a period of 60-72 hr, at either 4 O or 25O. The AChR is purified in Triton X-100 (see above), and this detergent is replaced by sodium cholate before the addition of lipids and dialysis. We do this either by centrifugation of AChR into a 5-20% sucrose gradient containing 0.5% sodium cholate or by repeated dilution with a 0.5% cholate and concentration against 0.5% cholate buffer in a collodion bag ultrafiltration apparatus (Schleicher and Schuell, Inc., Keene, N.H.). Lipids are isolated from Torpedo electric tissue by the procedure of Bligh and Dyer" and purified on a silica gel column using chloroform-methanol solutions for elution. The neutral lipids and the phospholipids (mainly phosphatidylethanolamine and phosphatidylcholine) are collected separately. A lipid mixture contaiing 50% egg phosphatidylcholine, 40% Torpedo phospholipids, and 10% Torpedo neutral lipids is evaporated to dryness under a stream of Nz and then suspended in buffer containing in different cases 0-3% sodium cholate. The lipid mixture is added to the AChR solution to give a 1ipid:protein weight ratio of 10: 1 and an AChR concentration of about 1 mg per ml. Routinely, traces of ["C]phosphatidylcholine and of [3H]MBTA-labeled receptor are included in the dialysis mixture to provide markers for the lipids and the AChR. This mixture is then dialyzed. A mixture containing only the lipids was usually dialyzed at the same time. The dialysis buffer is changed every 12 hr and usually contains 200 mM Na' (or K)-200 mM C1- (or 100 mM SOI'-)-lO mM Tris-0.5 mM EDTA-3 m M NaNs ( p H 7.4); 1-5 mM CaCL was included in some preparations. The binding properties of the receptor are unaffected by the dialysis procedure as judged by specific labeling with ['HJMBTA. We have shown previouslyE that AChR is incorporated into the vesicles by this procedure since the AChRphospholipid complex can be separated from free AChR and AChR-free phospholipid vesicles by flotation in a sucrose density gradient. The functional properties of the reconstituted vesicles are tested by measuring the rate of *'Na+ influx in the presence and absence of AChR agonists and antagonists. The vesicles are incubated at 2 5 O with *ma+,and at various times, 75 aliquots are applied to a 0.8-ml column of Bio-Gel P-6 equilibrated with isosmotic Tris-SO4 (pH 7.4). A fraction is collected at the void
Annals New York Academy of Sciences
180
100
80
-
"No
INFLUX
-
VALINOMYCIN ( l p p l l
0
40
20
60
FIGURE 3. Influx of "Na+ into reconstituted vesicles. Vesicles with and without Torpedo AChR were prepared as described in the text. To 150 pl of vesicles in 100 mM KSO.-lO mM Na2S04-10 mM Tris SO, (pH 7.4) was added 250 p l of the same buffer, 100 p1 of "Na+ (50 pCi in 110 mM K,S04) and 5 p1 of valinomycin (0.1 mg/ml in ethanol). At time t, 75-pl aliquots were passed through 0.8-ml Bio-Gel P-6columns equilibrated with isosmotic Tris SO, (pH 7.4). The vesicle fraction (0.5 ml) was counted in 5 ml Scintisol.
80
TIME (MIN)
volume within one minute that contains about 1 5 % of the vesicles and none of the free "Na'. The contained *'Na+ counts are divided by the counts due to the ["C]phospholipids, thus giving a normalized value for contained Na+ per weight of phospholipid. The vesicles in most cases are relatively impermeable to Na', having halftimes for equilibration of several hours at 2 5 O . The rate of influx can be increased substantially by adding gramicidin D or valinomycin ( 1 pg/ml), to the incubation mixture; however, little difference in permeability is seen between vesicles with and without AChR (FIGURE 3). By using mixtures of Na3S04 and KSO. in the dialysis and incubation buffers, we have been able to create transmembrane potentials across the vesicles, using valinomycin as a K-selective ionophore.In From results on intact ' I a potential difference (positive outside) might be expected to enhance receptor activity. We have not, however, observed any reproducible effect of AChR activators 22Na INFLUX 80
-
FIGURE 4. Effect of carbamylcoline (CARB) and valinomycin on "Na+ influx into AChR-lipid vesicles. The procedure is as described in FIGURE 3: 5 pl of 10-mM CARB and/or 5 p1 of valinomycin (0.1 mg/ml) were added at time zero to samples indicated to give final concentrations of 100 pM and 1 pg/ml, respectively.
2
0
0
20
40
TIME (MIN)
60
80
McNamee et al. : Purification of Acetylcholine Receptor
181
or inhibitors on the rate of "Na' influx either in the presence or absence of valinomycin (FIGURE4). Hazelbauer and Changeux' demonstrated that solubilization of AChR by cholate does not in itself destroy the permeability control properties of the AChR. Recently, Michaelson and Raftery' have reported that permeability control could sometimes be reconstituted by procedures similar to those described here, using purified Torpedo AChR and lipids. Our efforts to define the conditions necessary for the reconstitution of the permeability control function of the AChR in phospholipid membranes are continuing. Further characterization of solubilized receptor coupled with biophysical studies of receptor interactions in a membrane environment promise to provide information relevant to the mechanism of permeability control. REFERENCES
4.
KARLIN, A. 1974. Life Sci. 14: 1385-1415. RANG,H.P. 1974. Quart. Rev. Biophys. 7: 283-399. KARLIN, A., M. G. MCNAMEE, C. L. WEILL,& R. VALDERRAMA. In Methods in Receptor Research. M. Blecher, Ed. Marcel Dekker, Inc. New York, N.Y. In press. MICHAELSON, D. M. & M. A. RAFTERY. 1974. Proc. Nat. Acad. Sci. U.S.A.
5.
HAZELBAUER, G. L. & J.-P. CHANGEUX. 1974. Proc. Nat. Acad. Sci. U S A . 71:
6.
MCNAMEE, M. G., C. L. WEILL,& A. KARLIN.1975. In Protein-Ligand Interactions. H. Sund & G. Blauer, Eds. Verlag Walter de Gruyter. Berlin, Federal Republic of Germany. KARLIN, A. & D. A. COWBURN. 1973. Proc. Nat. Acad. Sci. USA. 12:
1. 2. 3.
71: 4768-4772.
1479-1483.
7.
363 6-3640. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
KARLSSON, E., H. ARNBERG & D. EAKER.1971. Eur. J. Biochem. 21: 1-16. BIESECKER, G. 1973. Biochemistry 12: 4403-4409. RICE,R. H. & G. E. MEANS.1971. J. Biol. Chern. 246: 831-832. BOROFF, D. A. & U . FLECK. 1966. J. Bact. 92: 1580-1581. WEILL,C. L., M. G. MCNAMEE, & A. KARLIN.1974. Biochem. Biophys. Res. Commun. 61: 997-1003. E. 1972. J. Biol. Chem. 247: 8198-8200. RACKER, BLIGH,E. G. & W. J. DYER.1959. Can. J. Biochem. Physiol. 37: 911-917. KORNBERG, R. D., M. G. MCNAMEE, & H. M. MCCONNELL. 1972. Proc. Nat. Acad. Sci. U.S.A. 69: 1508-1513. F. & H. GRUNDFEST. 1971. J. Gen. Physiol. 57: 71-92. RUIZ-MANRESA, MAGLEBY, K. L. & C. F. STEVENS. 1972. J. Physiol. Lond. 223: 173-197. LOWRY,0. H., N. J. ROSEBROUGH, A. L. FARR,& R. J. RANDALL. 1951. J. Biol. Chem. 193: 265-275. ELLMAN, G. L., K. D. COURTNEY, V. ANDRES, & R. M. FEATHERSTONE. 1961. Biochem. Pharm. 7: 88-95.
DISCUSSION DR. ELDEFRAWI:I think the experiments that you did with the ACh receptor of Torpedo are essentially a repetition of the work published by Michaelson
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Annals New York Academy of Sciences
and Raftery. They reported an increase in sodium efflux from the vesicles that were carrying receptor in response to applied carbamylcholine. Was your attempt similar to theirs then? Did your attempt fail, while they claimed at least partial success? DR. KARLIN:Yes, that is the case. Michaelson and Raftery have reported success, which was at least intermittent; very .few details are given so it’s hard to evaluate whether they got a specific or a general cation permeability. But if they succeeded, it’s very important to know whether this can work, even if it doesn’t succeed brilliantly or show a high increase in permeability, because we really want to know whether these receptors are complete and/or functional because none of the binding studies are assaying for the functional properties of the receptor. DR. TOSTESON: In the experiment in which valinomycin increased the sodium influx, were the K concentrations equal on the two sides? DR. KARLIN:Yes. DR. TOSTESON: Are the results the same? DR. KARLIN:Yes, but the kinetics are somewhat different. Interestingly as the field is dissipated, we see the influx reaching a peak and then coming down as if we are dissipating the gradient. In the absence of a gradient the influx approaches the same final value. DR. GOLDEN:By your gradient technique, when did you replace the Triton with cholate? Is there also a phospholipid equilibrated along the gradient? DR. KARLIN:No. DR. GOLDEN:I mention this because in recent work reported by Metcalfe in the Proceedings of the National Academy of Sciences they tried to change the lipid environment before reconstituting calcium ATPase. They found that in order to maintain pumping activity for subsequent reconstitution, phospholipid has to be equilibrated down the gradient. So perhaps if you have phospholipid when you’re replacing a detergent you might get some results. DR. KARLIN: We plan to do that.
Iv.
MECHANISM ACROSS SYNAPTICMEMBRANES
PERMEATION
PURIFICATION OF ACETYLCHOLINE RECEPTOR FROM TORPEDO CALIFORNICA AND ITS INCORPORATION INTO PHOSPHOLIPID VESICLES" Mark G. McNamee, Cheryl L. Weill, and Arthur Karlin Department of Neurology College of Physicians and Surgeons Columbia University N e w York, New York 10032
The acetylcholine receptors (AChR) translate the binding of acetylcholine into increases in ion permeabilities of synaptic membranes. The receptor protein can be solubilized from the membrane by nonionic detergents and purified by affinity chromatography. The isolated protein retains the specific binding properties attributed to the AChR in situ and is being used to deduce the molecular properties of the AChR. Purification and characterization of the AChR has been the subject of several recent reviews.'-3 A complete molecular description of receptor function must include its interactions with other membrane components. Considerable effort is now being dedicated to the reconstitution of cation permeability control in rnem branes containing AChR'-a and our initial efforts in this area are discussed.
PURIFICATION OF
ACETYLCHOLINE RECEPTOR
Tissue from the electric organ of Torpedo californica is homogenized in 1 mM EDTA (pH 7.4), and the pellet obtained after centrifugation is homogenized in 1 M NaCI-2 m M NaPOa-1 mM EDTA (pH 7.4), sedimented, and resuspended in 1 mM EDTA (pH 7.4), and resedimented. This crude membrane pellet is then extracted for 1 hr at 40 with 3% Triton X-100-50 mM NaCI-10 mM NaPO+-l mM EDTA-3 mM NaN3 (pH 8.0). The supernatant after centrifugation contains on the average 16% of the initial protein, 18% of the acetylcholinesterase ( AChE) , and nearly all of the AChR. The procedure used is identical to that used for extraction of AChR from Electrophorus electricus and complete details may be found elsewhere." The specific activity of the AChR in the detergent extract is approximately 300 pmol of sites per mg of protein, approximately ten times higher than that obtained with Electrophorus. Solubilized AChR is purified by affinity chromatography using p-carboxyphenyltrimethylammonium ion, coupled to Affi-Gel 401 (Bio-Rad Lab., Rich-
* This work was supported by Research Grants NS 07065 from the National Institute of Neurological Diseases and Stroke and BMS75-03026 from the National Science Foundation and by a grant-in-aid from the New York Heart Association, Incorporated. 175
176
Annals New York Academy of Sciences TABLE1 AFFINITY CHROMATOGRAPHIC PURIFICATION F R O M Torpedo californica
OF
RECEPTOR ~~~~~
Protein * Elution Sequence Extracts Input Not bound TNP5Oq TNPl 00 10 mM CARBII in TNP 90 Total recovery
mg 1133 900 6.9 8.5 26.3 942
AChEt
Receptor$
%
nmol
%
nmol
%
100 79.4 0.6
19.2 16.3 0.39 0.21 1.27 18.2
100 85 .O 2.0 1.1 6.6 95
371 0 0 1.5 73.5 75
100 0 0 0.6 19.8 20
0.8
2.3 83
*Protein was determined by the Lowry method with bovine serum albumin as the standard." t Acetylcholinesterase activity was measured by Ellman's p r o c e d ~ r e and ' ~ is expressed as nmoles of catalytic sites assuming 80,000 daltons per catalytic site and 10 mol acetylthiocholine hydrolyzed per min per g enzyme. $ Receptor activity was determined by specific [ 3H]MBTA labeling (see text). 8 Extract (186 ml) from 600 g of tissue in 3% Triton X-100-50 mM NaCl-10 mM NaP0,1 mM EDTA-3 mM NaN, (pH 7.0). 9 TNPxx contains 0.2% Triton X-100,xx mM NaCl, 10 mM NaPO,, 1 mM EDTA, 3 mM NaN, (pH 7.0). 11 Carbamylcholine was removed from fractions by dialysis before assay for AChE and receptor.
mond, Calif.), as the adsorbing ligand and carbamylcholine (CARB) as the eluting ligand. TABLE 1 provides a summary of a typical elution pattern from the affinity column. The AChR can be further purified about 30% by centrifugation in a 5-20% sucrose density gradient containing 0.2% Triton X-100.Typically, the AChR appears as two peaks (FIGURE 1 ) . The faster-moving peak moves as if it were a dimer of the slower peak. The specific activity of AChR is constant across both peaks at about 4 nmol of MBTA sites per mg of protein (see below). Both peaks are resolved from remaining traces of AChE and less than .001% of the pooled AChR fractions is active AChE. CHARACTERlZATION
OF
ACHR
Afinity Alkylation
The AChR can be assayed at all stages of purification by affinity alkylation of dithiothreitol-reduced receptor with 4-(N-maleimido)benzyltri-["H]-methy1ammonium iodide (['HIMBTA), using a modification of the "quick assay" procedure.' The portion of MBTA labeling that is blocked by the principal a-neurotoxin of Naja naja siamensis' is taken to be specific for AChR; all of the ['HIMBTA labeling of purified AChR is blocked by toxin.
McNamee et al. :
0
4
Purification of Acetylcholine Receptor
8
12
16
20
177
24
FRACTION
FIGURE 1. Sucrose density gradient centrifugation of Torpedo AChR. Acetylcholine receptor, purified by affinity chromatography ( 1 mg in 1 ml of TNPSO [see footnote (Q)to TABLE11, 3 nmol MBTA sites/mg) was applied to the top of a 16-1111 5-20% sucrose gradient in TNPlSO and centrifuged at 27,000 rprn for 16.5 hr at 5" in the SW27 rotor of an L2-65B Beckrnan ultracentrifuge. Fractions of 0.5 rnl were collected with an ISCO Model 183 Fractionator and analyzed for percentage of sucrose (-a+), protein (m, 10 pg/unit),I5 AChR ( 0 , 50 nmol MBTA sitedunit), and AChE (A,50 pmol/unit).a Approximately 90% of the protein, AChR, and AChE were recovered. The specific activity of the peak AChR fraction was 4.2 nmol MBTA siteslmg. Toxin Binding A ['Hlmethyl derivative of N . n. siamensis &-toxin was prepared by reductive alkylation of native toxin with formaldehyde and tritiated sodium borohydride,". lo The toxin is labeled to the extent of about 0.5 mol of methyl groups per mol of toxin, and yields about 2000 cpm per pmol under typical counting conditions. The biological activity of [3H]-toxin is indistinguishable from unlabeled toxin by a bioassay using micell and all of the ['HI-toxin is bound by an excess of receptor. The binding of ['HI-toxin to receptor is determined by incubating toxin and AChR for one hour at 2 5 O in 0.2% Triton X-100-100mM NaC1-10 mM NaP04-I mM EDTA-3 mM NaN, (pH 7.0) and then separating toxin-AChR complex from free toxin on Bio-Gel P-30. The bound radioactivity and the protein of the isolated complex are determined. We have found that the specific activity estimated by toxin binding is approximately twice that estimated by [3H]MBTA labeling. For example, a preparation of AChR containing 4.2 nmol of sites susceptible to ['HIMBTA labeling per mg of protein was found to bind 8.4 nmol of 3H-toxin per mg. The 2: 1 ratio does not appear to be dependent on the degree of purification. Since toxin blocks all of ["HIMBTA labeling of purified AChR, there must
178
Annals New York Academy of Sciences
be at least one interacting site of toxin and ['HIMBTA labeling. It has not yet been determined whether or not the additional toxin binding represents a second distinct binding site.
Subunit Structure AChR from Torpedo electric tissue purified by affinity chromatography displays two protein components by polyacrylamide gel electrophoresis under nondissociating conditions in the presence of Triton X-100 (FIGURE2A).
FIGURE2. Polyacrylamide gel electrophoresis of purified AChR. (A, C, and E) Triton X-100 Gels: To samples of AChR from the affinity column (A) and from a subsequent sucrose density gradient centrifugation, second peak (heavier) C and first peak (lighter) D was added solid sucrose to 10% and brornophenol blue. Samples containing 10-12 pg of proteins were applied to 3.6% gels, and run in 0.2% Triton X-100-100 mM Tris-acetate, pH 8 at 3 ma/gel. Note that gel A was not run as long as gels C and E (ink line marks front). (B, D, and F) Sodium dodecyl sulfate (SDS) gels: Samples of AChR obtained as above were dialyzed against 2% SDS-10 mM Tris acetate-2 mM EDTA-pH 8, precipitated, and washed with dry acetone to remove Triton X-100 and dissolved in an appropriate volume of dialysis buffer containing 20 mM DTT for 0.5-1 hr at SO" C. Samples containing 12-18 pg of protein were applied to 5.6% gels and run in 1% SDS-100 mM Tris-acetate, PI-I 8 at 8 ma/geI. Under dissociating conditions, saturated with sodium dodecyl sulfate (SDS) and reduced, this same material gives rise to four polypeptide bands corresponding to apparent molecular weights of 39,000, 48,000, 58,000, and 64,000 daltons (FIGURE 2B) .Iz Sucrose density gradient centrifugation results in the separation of two symmetrical peaks of protein and AChR activity, as described above. The protein from each peak yields a single band on Triton X-100 polyacryl2C and E ) . The relative mobilities of the single bands amide gels (FIGURE in C and D correspond to the relative mobilities of the bands in A. As expected, the dimer has a smaller electrophoretic mobility. The SDS gel pattern of the protein in each peak from the sucrose density gradient centrifugation (FIGURE2D and F) is identical to that obtained with AChR before sucrose density gradient centrifugation [FIGURE 2B). By SDS-gel electrophoresis, it has been shown that the affinity alkylation by 13H]MBTA is directed specifically and exclusively to the subunit of 39,000 daltons.'* This subunit thus bears all or part of the acetylcholine binding site, as previously demonstrated for the subunit of 40,000 daltons in AChR from Electrophorus.' The functional significance of the other components has yet to be ascertained. The specific activity of the most highly purified AChR preparations from
McNamee et al. : Purification of Acetylcholine Receptor
179
Torpedo (about 4 nmol MBTA sites per mg protein) corresponds to a minimal molecular weight of 250,000 daltons per MBTA binding site. The combined apparent molecular weights of the four putative subunits is 209,000.
INCORPORATION
OF
ACHR
INTO
PHOSPHOLIPID VESICLES
Reconstitution of AChR function in model membrane systems is a principal objective of current receptor research. The mechanism by which the binding of acetylcholine and its congeners to the AChR is transduced into a change in membrane cation permeability is not known, and reconstituted vesicles offer the most attractive system for investigating permeability control at the molecular level. We incorporate purified AChR into phospholipid vesicles using a dialysis procedure similar to that used by Racker"' for a variety of membrane systems. The vesicles are formed by dialyzing a solution of AChR and lipids in sodium cholate against detergent-free buffer, under N2, during a period of 60-72 hr, at either 4 O or 25O. The AChR is purified in Triton X-100 (see above), and this detergent is replaced by sodium cholate before the addition of lipids and dialysis. We do this either by centrifugation of AChR into a 5-20% sucrose gradient containing 0.5% sodium cholate or by repeated dilution with a 0.5% cholate and concentration against 0.5% cholate buffer in a collodion bag ultrafiltration apparatus (Schleicher and Schuell, Inc., Keene, N.H.). Lipids are isolated from Torpedo electric tissue by the procedure of Bligh and Dyer" and purified on a silica gel column using chloroform-methanol solutions for elution. The neutral lipids and the phospholipids (mainly phosphatidylethanolamine and phosphatidylcholine) are collected separately. A lipid mixture contaiing 50% egg phosphatidylcholine, 40% Torpedo phospholipids, and 10% Torpedo neutral lipids is evaporated to dryness under a stream of Nz and then suspended in buffer containing in different cases 0-3% sodium cholate. The lipid mixture is added to the AChR solution to give a 1ipid:protein weight ratio of 10: 1 and an AChR concentration of about 1 mg per ml. Routinely, traces of ["C]phosphatidylcholine and of [3H]MBTA-labeled receptor are included in the dialysis mixture to provide markers for the lipids and the AChR. This mixture is then dialyzed. A mixture containing only the lipids was usually dialyzed at the same time. The dialysis buffer is changed every 12 hr and usually contains 200 mM Na' (or K)-200 mM C1- (or 100 mM SOI'-)-lO mM Tris-0.5 mM EDTA-3 m M NaNs ( p H 7.4); 1-5 mM CaCL was included in some preparations. The binding properties of the receptor are unaffected by the dialysis procedure as judged by specific labeling with ['HJMBTA. We have shown previouslyE that AChR is incorporated into the vesicles by this procedure since the AChRphospholipid complex can be separated from free AChR and AChR-free phospholipid vesicles by flotation in a sucrose density gradient. The functional properties of the reconstituted vesicles are tested by measuring the rate of *'Na+ influx in the presence and absence of AChR agonists and antagonists. The vesicles are incubated at 2 5 O with *ma+,and at various times, 75 aliquots are applied to a 0.8-ml column of Bio-Gel P-6 equilibrated with isosmotic Tris-SO4 (pH 7.4). A fraction is collected at the void
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FIGURE 3. Influx of "Na+ into reconstituted vesicles. Vesicles with and without Torpedo AChR were prepared as described in the text. To 150 pl of vesicles in 100 mM KSO.-lO mM Na2S04-10 mM Tris SO, (pH 7.4) was added 250 p l of the same buffer, 100 p1 of "Na+ (50 pCi in 110 mM K,S04) and 5 p1 of valinomycin (0.1 mg/ml in ethanol). At time t, 75-pl aliquots were passed through 0.8-ml Bio-Gel P-6columns equilibrated with isosmotic Tris SO, (pH 7.4). The vesicle fraction (0.5 ml) was counted in 5 ml Scintisol.
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volume within one minute that contains about 1 5 % of the vesicles and none of the free "Na'. The contained *'Na+ counts are divided by the counts due to the ["C]phospholipids, thus giving a normalized value for contained Na+ per weight of phospholipid. The vesicles in most cases are relatively impermeable to Na', having halftimes for equilibration of several hours at 2 5 O . The rate of influx can be increased substantially by adding gramicidin D or valinomycin ( 1 pg/ml), to the incubation mixture; however, little difference in permeability is seen between vesicles with and without AChR (FIGURE 3). By using mixtures of Na3S04 and KSO. in the dialysis and incubation buffers, we have been able to create transmembrane potentials across the vesicles, using valinomycin as a K-selective ionophore.In From results on intact ' I a potential difference (positive outside) might be expected to enhance receptor activity. We have not, however, observed any reproducible effect of AChR activators 22Na INFLUX 80
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FIGURE 4. Effect of carbamylcoline (CARB) and valinomycin on "Na+ influx into AChR-lipid vesicles. The procedure is as described in FIGURE 3: 5 pl of 10-mM CARB and/or 5 p1 of valinomycin (0.1 mg/ml) were added at time zero to samples indicated to give final concentrations of 100 pM and 1 pg/ml, respectively.
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or inhibitors on the rate of "Na' influx either in the presence or absence of valinomycin (FIGURE4). Hazelbauer and Changeux' demonstrated that solubilization of AChR by cholate does not in itself destroy the permeability control properties of the AChR. Recently, Michaelson and Raftery' have reported that permeability control could sometimes be reconstituted by procedures similar to those described here, using purified Torpedo AChR and lipids. Our efforts to define the conditions necessary for the reconstitution of the permeability control function of the AChR in phospholipid membranes are continuing. Further characterization of solubilized receptor coupled with biophysical studies of receptor interactions in a membrane environment promise to provide information relevant to the mechanism of permeability control. REFERENCES
4.
KARLIN, A. 1974. Life Sci. 14: 1385-1415. RANG,H.P. 1974. Quart. Rev. Biophys. 7: 283-399. KARLIN, A., M. G. MCNAMEE, C. L. WEILL,& R. VALDERRAMA. In Methods in Receptor Research. M. Blecher, Ed. Marcel Dekker, Inc. New York, N.Y. In press. MICHAELSON, D. M. & M. A. RAFTERY. 1974. Proc. Nat. Acad. Sci. U.S.A.
5.
HAZELBAUER, G. L. & J.-P. CHANGEUX. 1974. Proc. Nat. Acad. Sci. U S A . 71:
6.
MCNAMEE, M. G., C. L. WEILL,& A. KARLIN.1975. In Protein-Ligand Interactions. H. Sund & G. Blauer, Eds. Verlag Walter de Gruyter. Berlin, Federal Republic of Germany. KARLIN, A. & D. A. COWBURN. 1973. Proc. Nat. Acad. Sci. USA. 12:
1. 2. 3.
71: 4768-4772.
1479-1483.
7.
363 6-3640. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
KARLSSON, E., H. ARNBERG & D. EAKER.1971. Eur. J. Biochem. 21: 1-16. BIESECKER, G. 1973. Biochemistry 12: 4403-4409. RICE,R. H. & G. E. MEANS.1971. J. Biol. Chern. 246: 831-832. BOROFF, D. A. & U . FLECK. 1966. J. Bact. 92: 1580-1581. WEILL,C. L., M. G. MCNAMEE, & A. KARLIN.1974. Biochem. Biophys. Res. Commun. 61: 997-1003. E. 1972. J. Biol. Chem. 247: 8198-8200. RACKER, BLIGH,E. G. & W. J. DYER.1959. Can. J. Biochem. Physiol. 37: 911-917. KORNBERG, R. D., M. G. MCNAMEE, & H. M. MCCONNELL. 1972. Proc. Nat. Acad. Sci. U.S.A. 69: 1508-1513. F. & H. GRUNDFEST. 1971. J. Gen. Physiol. 57: 71-92. RUIZ-MANRESA, MAGLEBY, K. L. & C. F. STEVENS. 1972. J. Physiol. Lond. 223: 173-197. LOWRY,0. H., N. J. ROSEBROUGH, A. L. FARR,& R. J. RANDALL. 1951. J. Biol. Chem. 193: 265-275. ELLMAN, G. L., K. D. COURTNEY, V. ANDRES, & R. M. FEATHERSTONE. 1961. Biochem. Pharm. 7: 88-95.
DISCUSSION DR. ELDEFRAWI:I think the experiments that you did with the ACh receptor of Torpedo are essentially a repetition of the work published by Michaelson
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and Raftery. They reported an increase in sodium efflux from the vesicles that were carrying receptor in response to applied carbamylcholine. Was your attempt similar to theirs then? Did your attempt fail, while they claimed at least partial success? DR. KARLIN:Yes, that is the case. Michaelson and Raftery have reported success, which was at least intermittent; very .few details are given so it’s hard to evaluate whether they got a specific or a general cation permeability. But if they succeeded, it’s very important to know whether this can work, even if it doesn’t succeed brilliantly or show a high increase in permeability, because we really want to know whether these receptors are complete and/or functional because none of the binding studies are assaying for the functional properties of the receptor. DR. TOSTESON: In the experiment in which valinomycin increased the sodium influx, were the K concentrations equal on the two sides? DR. KARLIN:Yes. DR. TOSTESON: Are the results the same? DR. KARLIN:Yes, but the kinetics are somewhat different. Interestingly as the field is dissipated, we see the influx reaching a peak and then coming down as if we are dissipating the gradient. In the absence of a gradient the influx approaches the same final value. DR. GOLDEN:By your gradient technique, when did you replace the Triton with cholate? Is there also a phospholipid equilibrated along the gradient? DR. KARLIN:No. DR. GOLDEN:I mention this because in recent work reported by Metcalfe in the Proceedings of the National Academy of Sciences they tried to change the lipid environment before reconstituting calcium ATPase. They found that in order to maintain pumping activity for subsequent reconstitution, phospholipid has to be equilibrated down the gradient. So perhaps if you have phospholipid when you’re replacing a detergent you might get some results. DR. KARLIN: We plan to do that.
