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bilayers by the septum-supported, vesicle-derived bilayer technique, which has been reviewed by Schindler elsewhere in this series.37 In the planar bilayer configuration, the ion conductance activity of purified calcium channels is modulated by calcium channel activators and inhibitors as expected. Activation of the channel by Bay K8644 required phosphorylation by cAMP-dependent protein kinase, and the level of channel activity was greatly increased by phosphorylation as observed in ion flux experiments. However, in the planar bilayer system, individual ion conductance events ranged in size from 0.9 to 60 pS, in contrast to the 20-pS values observed with patch pipette methods. Nevertheless, the results of ion flux, patch pipette, and macroscopic bilayer methods together show that purified skeletal muscle calcium channels retain selective ion conductance activity that is modulated appropriately by calcium channel-modulating drugs and protein phosphorylation. 37 H. Schindler, this series, Vol 171, p. 225.
[36] P u r i f i c a t i o n a n d R e c o n s t i t u t i o n o f N i c o t i n i c Acetylcholine Receptor
By AMITABHCHAK and ARTHUR KARLIN Introduction The nicotinic acetylcholine receptor (AChR) was the first receptor, and certainly the first ligand-gated channel, to be purified (see Ref. 1 for review). This success during 1973 and 1974 was due largely to the availability of very rich sources, namely, the electric tissues of electric rays (family Torpedinidae) and the electric eel (Electrophorus electricus), and to the availability of reversible and irreversible ligands for the acetylcholine binding sites. Also, at that time there was increasing awareness of the properties of amphipathic, integral membrane proteins, of the hydrophobic effect, and of detergents.2 It became clear that the techniques used to purify water-soluble proteins could be applied to membrane proteins in detergent complexes. The minimal criteria for the choice of detergent were that the protein was efficiently solubilized and that at least one assayable property, binding in the case of receptors, was preserved. For receptors like the AChR that are ligand-gated channels, although 1A. Karlin, in "The Cell Surface and Neuronal Function" (C. W. Cotman, G. Poste, and G. L. Nicolson, eds.), p. 191. Elsevier/North-Holland Biomedical, Amsterdam, 1980. 2 C. Tanford, "The Hydrophobic Effect," 2nd Ed. Wiley, New York, 1980.
METHODS IN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPrem,Inc. Allrightsofreproductlonin any form re,fred,
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binding is preserved in detergent solution, the critical functional properties of gating and ion conduction are observed only in membranes. Therefore to assay these functions in purified ligand-gated channels, one must reincorporate them into artificial membranes. Also, studies of receptor topology and interactions with other membrane proteins and lipids require either native membranes or reconstituted membranes. We describe the preparation, starting with Torpedo californica electric tissue, of AChR-rich native membranes, solubilized and purified AChR, and AChR reconstituted in liposomes. Acetycholine receptor subtypes have also been purified from other tissues (e.g., muscle and neuronal tissue) and from many other species (see Ref. 3 for review). Recently, many subtypes have been characterized genetically rather than biochemically (see Ref. 4 for review). Electric Tissue Live rays5 are dissected, and the tissue is stored in liquid nitrogen. Alternatively, tissue can be purchased already frozen. In the former case, working in the cold room and wearing rubber gloves, we remove the ray from its container and immediately pith it. The two electric organs fill the "wings." A scalpel is used to cut around the edges of the kidney-shaped organs. The dermal layer on the dorsal side is peeled off with a surgical damp, following which the organ is shelled offthe ventral surface by blunt dissection. The electric organs are weighed, placed on an enameled metal tray, and cut into approximately 2 cm chunks, which are dropped one by one into liquid nitrogen for quick freezing. We have stored tissue under liquid nitrogen for several months. A medium-sized ray yields 600 to 1200 g of electric tissue. Preparation of M e m b r a n e s Rich in Nicotinic Acetylcholine R e c e p t o r For the preparation of AChR-rich membranes, ~ approximately 120 g of frozen Torpedo tissue is thawed in 300 ml of PE buffer.7 Subsequent 3j. Lindstrom,R. Schoepfer,and P. Whiting,Mol. Neurobiol. l, 281 (1987). 4T. Claudio, in "Frontiers of Neurobiology: Molecular Neurobiology"(D. Glover and D. Haines,eds.),p. 63. IRL, London, 1989. 5PacificBiomarine(Venice,CA) or Marinus(LongBeach,CA). 6C. Czajkowski,M. DiPaola, M. Bodkin,G. Salazar-Jimenez,E. Holtzman,and A. Karlin, Arch. Biochem. Biophys. 272, 412 (1989). 7Abbreviationsfor solutions: PE bufferis l0 mM sodiumphosphate, 1 mM EDTA, pH 7; NPxxx is xxx mM NaCl, l0 mM sodiumphosphate, 1 mM EDTA, 0.02% NAN3,pH 7.0, unless otherwisespecifiedin parentheses,as, for example,NP100 (pH 8.0); TNPxxxis 0.2% Triton X-100 in NPxxx; 2CNPxxx(pH 7.4) is 2% sodiumcholatein NPxxx(pH 7.4); 0.5 CNPxxx(pH 7.4) is 0.5%sodiumcholatein NPxxx(pH 7.4); B bufferis 0.2%TritonX-100, 10 mM NaC1, l0 mM MOPS, pH 7.4.
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operations are carried out at 4 ° . The mixture containing nearly thawed tissue is placed in a food processor 8 and minced by four bursts of 30 sec each. Most of the foam is allowed to settle. One-third of the suspension at a time is homogenized at full speed, twice, for 1 min, in a 500-ml flask of a homogenizer9 fitted with two sharpened blades. The resulting suspension (H1) is centrifuged in two JA-14 rotor bottles 1° at 6000 rpm for 10 min. The supernatants are pooled and saved on ice. The pellets are suspended in I00 ml of PE buffer and rehomogenized in a 250-ml flask,9 at full speed, twice, for 1 min. This supernatant is added to the saved pool, which is then filtered through eight layers of cheesecloth. The pellets (PI) are discarded. The supernatant (S1) is centrifuged in 26-ml polycarbonate bottles in a 60 Ti rotor 1° at 45,000 rpm for 40 min. The resulting supernatant ($2) is discarded. The pellets (P2) are suspended in 100 ml of 40% (w/v) sucrose in PE buffer and homogenized in a 250-ml flask at full speed twice for 1 min. This homogenate (H2) is centrifuged in polycarbonate bottles in a 60 Ti rotor at 60,000 rpm for 140 min. The resulting supernatants ($3) are discarded. The pellets (P3) are suspended in 40 ml PE buffer and homogenized in a 100-ml flask, 9 with one blade on the shaft, at full speed for l min. Homogenized P3 is frozen and stored in liquid nitrogen in 4-ml aliquots. Discontinuous sucrose density gradients with 12 ml of 44% (w/v) sucrose in PE buffer under l0 ml of 40% (w/v) sucrose are made in 60 Ti rotor polycarbonate bottles. P3 is removed from liquid nitrogen and thawed. Eighteen milliliters of P3 is mixed with 3.6 ml of 3 M NaCI in PE buffer to give a final concentration of 0.5 M NaC1. This suspension is passed in and out l0 times through a blunted 22-gauge, 3.5-inch spinal needle attached to a 30-ml plastic syringe. A 3-ml aliquot of this suspension (H3) is layered over each of six discontinuous gradients, and the gradients are centrifuged in a 60 Ti rotor at 60,000 rpm for 140 min. After centrifugation, two bands and a pellet are visible. The second band at the interface of the 40 and 44% sucrose layers is collected by suction from the top. These second bands are pooled and diluted with 4 to 5 volumes of PE buffer to a volume of 150 ml and centrifuged at 60,000 rpm in a 60 Ti rotor for 80 min. The resulting pellets in 4 ml of PE buffer are passed in and out, 10 times, through a 22-gauge needle, and this suspended AChRrich membrane is stored in liquid nitrogen. The yield is 1 to 2 mg of protein at a speofic activity of about 3 nmol of a-bungarotoxin binding sites per milligram of protein. The protein of this membrane therefore is about 40% AChR. Further purification can be s Cuisinart Model DLC-10 Plus. 9 Homogenizer Model Virtis-45, The Virtis Co., Inc. (Garcliner, NY). ~0 Rotors and centrifuges from Beckman Instruments Inc. (Palo Alto, CA).
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obtained by extraction o f peripheral m e m b r a n e proteins with dilute N a O H ( p H 1 1) ~t or with lithium diiodosalicylate, ~2but with consequent increased disordering o f the membrane. Also, affinity partitioning has given high specific activities.~3 Purification of Detergent-Solubilized Receptor The detergent used to solubilize the AChR should give a good yield while maintaining structural and functional integrity. Triton X-100 efficiently solubilizes the A C h R and maintains its snake curarimimefic toxinbinding activity and, with some alteration, its binding o f acetylcholine (ACh) and other small ligands.~ Ligand-gated channel activity o f the AChR cannot be recovered once it has been solubilized in Triton X-100, even though the solubilized A C h R can be reincorporated into liposomes) 4 For reconstitution of channel activity, solubilization and purification in cholate and phospholipid mixtures are required) 5 For structural studies, the higher yield and purity obtained by purification in Triton X-100 is preferred. We describe solubilization and purification o f AChR both in Triton X-100 and in a cholate and phospholipid mixture.
Preparation of Affinity Gel Bromoacetylcholine Bromide (BAC) ~6. T o 18.4 g o f choline bromide is added dropwise, over 40 rain, with stirring, 24.2 g o f b r o m o a c e t y l bromide. The viscous mixture is stirred for an additional 75 rain in an ice bath. T o the mixture is slowly added 75 ml o f absolute ethanol. The white crystalline product is recrystallized twice from 400 ml o f 2-propanol. The yield is 67% and the product melts at 135 o _ 136 °. BAC is stored desiccated at 4 ° and is not opened for weighing until at r o o m temperature. Affinity geP 7. Affi-Gel 10, ~s 25 ml in isopropanol as supplied, is washed 1~R. R. Neubig, E. K. Krodel, N. D. Boyd, and J. B. Cohen, Proc. Nat. Acad. S¢i. U.S.A. 76, 690 (1979). ~2j. Elliot, S. G. Blanehard, W. Wu, J. Miller, C. D. Strader, P. Hartig, H.-P. Moore, J. Rats, and M. A. Rat~ery,Biochem. J. 185, 667 (1980). t3 G. Johansson, R. Gysin, and S. D. Flanagan, a*.Biol. Chem. 256, 9126 (1981). ~4M. G. MeNamee, C. L. Weill, and A. Karfin, in "Protein-Lip,and Interactions" (H. Sund and G. Blauer, eds.), p. 316. deGruyter, Berlin, 1975. ~5M. Epstein and E. Racker, J. Biol. Chem. 253, 6660 (1978). ~6V. N. Damle, M. MeLaughlin, and A. Karlin, Biochem. Biophys. Res. Commun. 84, 845 (1978). t7 We previously used Afli-Gel401, TM a crosslinked agarose gel with attached homocysteinyl residues at about 10/tmol per ml packed gel, which we reacted with BAC to form the cholinocarboxymethyl adduct [A. Reynolds and A. Karlin, Biochem. 17, 2035 (1978)]. Bio-Rad recently discontinued manufacture of Atii-Gel 401. We now use Afli-Gel 10, a N-hydroxysuccinimideester linked to crosslinked agarose by a neutral 10 atom spacer. is Bio-Rad Laboratories, Inc. (Richmond, CA).
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PURIHCATION OF ION CHANNEL PROTEINS AND GENES
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in a Buchner funnel with 250 ml of cold water. The gel should not be allowed to dry during this or any subsequent step. The wet gel is transferred to a beaker with 75 ml 13.3 mMcystamine in 100 m M HEPES (pH 8.0). This slurry is shaken for 1 hr at room temperature. The gel is transferred to a Buchner and washed with 50 ml HEPES buffer and 4 × 50 ml water. The gel is mixed in a beaker with 50 ml 20 m M dithiothreitol in 200 m M TRIS buffer (pH 8.0) and shaken for 1 hr. The gel is washed with 600 ml water. Aliquots (100/zl) of the gel and of the supernatant are saved for sulfhydryl assay. The gel is mixed with 50 ml 20 m M BAC in 200 m M NaC1, 50 m M sodium phosphate buffer (pH 7.0) and shaken for 1 hr. The gel is washed with 500 ml water. Before the reaction with BAC, the gel contains about 10/zmol sulfhydryl per ml gel, and after BAC it contains no free sulfhydryls. The affinity gel is made before the membrane extract. The gel can be stored at 4 ° in 0.02% NaN 3 for a few days. Assayfor Sulfhydryl Groups in Gel. A gel sample is pipetted into a tared 12-ml conical glass centrifuge tube and sedimented for 1 min. The supernatant is carefully removed with a pulled out Pasteur pipette, and the packed gel is weighed. The gel is suspended by vortexing in 1 ml of 1 m M 5,5'-dithiobis(2-nitrobenzoic acid) in 200 m M Tris-C1 (pH 8.0) and after 1 min is sedimented. To 100/zl of the supernatant is added 1 ml of 200 m M Tris-C1 (pH 8.0), and the absorbance at 412 nm of this mixture is determined. A control without gel is treated similarly. A molar extinction coefficient of 13,600 is used to determine the sulfhydryl concentration in the cuvette, from which the sulfhydryl titer of the gel can be calculated. If after treatment with BAC the gel contains unreacted sulfhydryl groups, these can be blocked with N-ethylmaleimide.
Crude Membrane Preparation For the preparation of crude membranes, 19 600 g of Torpedo tissue is partially thawed and added to 2400 ml of 1 m M EDTA, pH 7.4. (If preservation of Torpedo AChR dimers is desired, 2 m M N-ethylmaleimide is added here to block free sulfhydryls, preventing the reduction of the disulfide linking the monomers in dimer.) All subsequent steps are carried out at about 4*. Two milliliters of 0.5 M phenylmethylsulfonyl fluoride (PMSF) in ethanol is added, and the tissue is homogenized for 60 sec in a 1-gallon commercial blender a° set at "low." This homogenate is filtered through four layers of cheesecloth and centrifuged in about 10 JA-14 polycarbonate bottles at 14,000 rpm for 90 rain. The supernatant is discarded. The pellets are collected in 1200 ml of the EDTA buffer and 19 Co L. WeiU, M. G. McNamee, and A. Karlin, Biochem. Biophys. Res. Commun. 61, 997 (1974). 20 Waring Products Division (New Hartford, CT).
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homogenized in the blender set at "high" for 20 see. This homogenate is centrifuged in the JA-14 rotor at 14,000 rpm for 90 rain. The supernatant is removed carefully from the loose pellets and discarded. The pellets are pooled in a 250-ml graduated cylinder and brought up to a volume of 173 ml with EDTA buffer; 21 ml of 500 m M NaC1, 100 m M sodium phosphate, l0 m M EDTA, 0.2% sodium azide, pH 7, is added. This suspension is homogenized in the l-liter container for the blender set at "medium" for 20 sec.
Extraction of Membrane in Triton X-I O0 For Triton X-100 extraction, 19 the crude membrane preparation is stirred vigorously in a 250-ml Erlenmeyer flask, while 50/21 of 0.5 M P M S F in ethanol and 16 ml of 40% Triton X-100 in water is added. The final medium contains 3% Triton X-100, 50 m M NaCI, 10 m M sodium phosphate, 1 m M EDTA, 0.02% azide pH 7. After being stirred for 1 hr at 4 °, the mixture is transferred to eight 60 Ti rotor tubes and centrifuged at 60,000 rpm for 30 rain. The extract is carefully removed from the pellets and pooled. It can be stored overnight in ice and in the refrigerator, or it can be loaded slowly onto the affinity column, for elution the next day.
Affinity Chromatography in Triton X-IO0 The extract can be adsorbed onto the affinity gel either prior to pouting the column or after pouring the column. '7,19 All operations are carried out in a cold room. In the first case, 24 ml of affinity gel is washed 3 times with 25 ml of TNP50. 7 The affinity gel and extract are stirred together with a magnetic bar at slow speed in a 250-ml beaker for 2 hr. The suspension is transferred to four 50-ml centrifuge tubes and sedimented for 4 rain. The supernatant is carefully removed. The gel is washed twice with TNP50. About 25 ml of TNP50 is added to the gel, and the slurry is transferred with a broken-off Pasteur pipette into a 1.5-era diameter glass column, using more TNP50 to complete the transfer as needed. During this process, eluate is collected at 1 drop]see at a drop size of 15 #1. In the second ease, the affinity gel is poured into a column in TNP50. The extract is loaded onto this column overnight at a flow rate of 10 to 15 ml/hr, controlled by a peristaltic pump. After the extract is loaded, the column is washed with 200 ml of TNP50 at 60 ml/min. After loading the extract by either method, the column is washed with 200 ml of TNP100 at 60 ml/hr. Finally, the AChR is eluted with 50 ml of 10 m M earbamylcholine in TNP90 at 30 ml/hr. This eluate is collected in 5-ml fractions, and the AChR elutes in fractions 2 through 7. These fractions in 1-ml aliquots are frozen in liquid nitrogen for assay and use. A
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typical yield is about 30 mg of AChR with specific activities in the different fractions ranging from 5 to 8 nmol curarimimetic toxin binding sites per milligram of protein. (The theoretical maximum specific activity is 7 nmol sites/mg, but there are inaccuracies in both the binding site and protein assays.) The above procedure results in pure or nearly pure AChR from Torpedo californica. In variations of the above method, slight additional purification and, in addition, resolution of monomeric and dimeric forms of AChR were obtained by sucrose density gradient centrifugation? TM In purifying AChR from less concentrated sources than Torpedo, other laboratories have used, in addition to affinity chromatography, chromatography on DEAE-cellulose, 22 on immobilized lectin, 23 and on immobilized antibody. ~ Also, affinity chromatography has been carried out on agarose linked either to other small ligands 21 or to various elapid snake toxins. 25
Purification in Cholate and Phospholipid Mixture The following variations of the above procedures preserve the ligandgated channel function of the AChR, which one can assay on reincorporation of the AChR into membrane. 26 A crude membrane fraction is prepared as above, up to the last sedimentation, except typically at one-sixth the scale, starting with 100 g of tissue. The l-liter blender container is used in place of the 1-gallon container. The crude membrane pellet is suspended in a final volume of 60 ml of 0.5% asolectin 27 in 2CNP100 (pH 7.4) 7 by homogenization at "low" for 20 sec. The mixture is stirred for 1 hr and then centrifuged at 60,000 rpm for 30 min. The supernatant is used immediately or stored in ice overnight. The BAC-Affi-Gel 10 affinity gel described above is washed with 0.0625% asolectin in 0.5CNP50 (pH 7.4), and 6 ml is poured into a 0.9-cm-diameter glass column. The supernatant is pumped onto the column at 30 ml/hr. The column is washed at the same rate successively with 25 ml of 0.5CNP50 (pH 7.4) and 50 ml of 0.5CNP100 (pH 7.4), and the AChR is eluted with 15 ml of 10 m M carbamylcholine in 0.5CNP90 (pH 7.4). The elution of protein can be followed in this case by its absorbance at 280 nm. Carbamylcholine-eluted fractions containing appreciable protein are stored in liquid nitrogen. 21 A. Karlin, M. G. McNamee, C. L. Weill, and R. Valderrama, in "Methods in Receptor Research" (M. BLecher, ed.), p. 1. Dekker, New York, 1976. 22 G. Biesecker, Biochemistry 12, 4403 (1973). 23 S. Froehner, C. G. Reiness, and Z. Hall, J. Biol. Chem. 252, 8589 (1977). u p. j. Whiting and J. Lindstrom, Biochemistry 25, 2082 (1986). 25 j. Lindstrom, B. Einarson, and S. Tzartos, this series, Vol. 74, p. 452. 26 R. L. Huganir, M. A. ScheU, and E. Racker, FEBSLett. 108, 155 (1979). 27 Associated Concentrates (Woodside, NY).
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Assays Acetylcholine Binding Sites Elapid snake venom contains basic polypeptide toxins which bind with high specificity and affinity to the ACh binding sites. Radioactive derivatives of these toxins are available. For example, the widely used a-bungarotoxin (Bgtx) is commercially available as an ~25I derivative,28 and Naja naja siamensis toxin 3 is readily tritiated. 29 In the former case, the iodinated toxin (initially about 140 Ci/mmol) is diluted 50 to 100 times with unlabeled ~-bungarotoxin 3° prior to use. 3~ We henceforth refer to the high-specific activity ~25I-labeledbungarotoxin diluted with unlabeled Bgtx simply as [125I]Bgtx. On the day of use, [~2SI]Bgtx is diluted to about 140 pmol/ml in 100 m M NaC1, 10 m M MOPS, pH 7.4, 0.2% Triton X-100, and 0.1 mg/ml bovine serum albumin. Two 25-mm DE-81 filters32 are placed on each screen of a filtration manifold and are washed with 4 ml of buffer B. Fifty microliters of diluted [~25I]Bgtxis added to 50/tl of AChR containing about 2 pmol of sites and mixed well. After 30 rnin at room temperature, the mixture is diluted with 5 ml of cold B buffer7 and poured onto the doubled DE-81 filters. 29,33 Mild suction at 50 to 100 m m Hg pressure difference is applied, the tube is washed onto the filter with another 5 ml of B buffer, and the filter is washed with 15 ml more of B buffer, now with a pressure difference of 200 m m Hg for faster filtration. After the filters are placed in the bottom of a scintillation vial, 250/tl of 1 M acetic acid is pipetted onto 2s New England Nuclear Corp. (Boston, MA). 29 V. N. Damle and A. Karlin, Biochemistry 17, 2039 (1978). 3o Sigma Chemical Co. (St. Louis, MO). 31 One should determine that [~2sI]Bgtx and Bgtx bind equivalently to AChR as follows. A fixed quantity of AChR is titrated with the diluted [~2~I]Bgtx.If uniabeled Bgtx bound with higher affinity than {125I]Bgtx,then the quantity of 12sIbound would reach a maximum at equivalence, when all Bgtx plus [125I]Bgtx were bound and all toxin binding rites were occupied, and then the 125Ibound would dc~'rea~ with increasing total Bgtx concentration to a new steady level as the excess unlabeled Bgtx competed with the [1251]BgUtfor the toxin binding sites; that is, there would be a cusp in the binding curve around equivalence. If no cusp is observed, one may conclude that labeled and unlabeled Bgtx bind equivalently for the purposes of tbis assay. In addition, one should routinely determine the fraction of t25Iin the [12SI]Bgtx preparation that is bound by excess AChR. This fraction is typically about 85%. One determines the factor (cpm/mol) for converting radioactivity to the quantity of Bgtx by counting a known quantity of [t25I]Bgtx plus Bgtx on filters, by multiplying these counts by the fraction of |2sI that could be bound by AChR, and by dividing by the quantity of Bgtx plus [1251]Bgtxin the sample. 32 Whatman, Inc. (Clifton, N J). 33 R. P. Klett, B. W. Fulpius, D. Cooper, M. Smith, E. Reich, and L. D. Possam, J. Biol. Chem. 252, 4811 (1973).
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the filters. After I 0 to 20 min at room temperature, 5 ml of Scintisol~ or equivalent counting cocktail is added, and the vial is placed in a scintillation counter.
Composition by Sodium Dodecyl Sulfate Gel Electrophoresis Standard Laemmli a5 conditions are used with a 7.5% acrylamide resolving gel. The sample is prepared initially by dissolving in 2% sodium dodecyl sulfate (SDS), 10 m M Tris, pH 8.0, 10 m M DTT, and holding at 50 ° for 30 min. (Higher temperatures can lead to degradation ofsubunits.) Thirty millimolar N-ethylmaleimide is added, and after an additional 15 min 9 volumes of acetone is added. After a few minutes, the precipitate is sedimented in a clinical centrifuge and washed with 5 volumes of acetone. The pellet is dried in a vacuum desiccator and dissolved in Laemmli sample buffer. Reduced and alkylated AChR yields four bands (a, fl, 7, and 6) with apparent masses of about 40, 48, 58, and 64 kDa? 9 If DTT and 2-mercapto-ethanol in the Laemmli sample buffer are omitted, the 64-kDa band (6) is diminished, and a 130-kDa band (~ dimer) is seen instead. Purity is indicated by the absence of other bands on the gel.
Reconstitution and Flux A s s a y 15,36,37 For reconstitution and flux assays,~mt,37 ligand-gated channel activity is more effectively reconstituted in a mixture of phosphatidylethanolamine (PE), 3s phosphatidylserine (PS), 3s and cholesterol than in asolectin. A P E P S - cholesterol mixture, 6: 3: 1 by weight, is made at 100 mg/ml in chloroform. Two hundred microliters of this mixture is dried to a film with a stream of N2 and then dried further under reduced pressure for 3 hr. To the dried lipid is added 570/zl of 2CNPI00 (pH 8.0). 7 The final concentration of lipid is 3.5%. The mixture is vortexed and sonicated under N2 in a bath sonicator 39 until the mixture is opalescent (about 5 min). Native Torpedo membrane vesicles are quite leaky. The ligand-gated channel activity of the AChR can be more reproducibly assayed after solubilization and reconstitution into liposomes. Membrane, containing 34Isolab, Inc. (Akron, OH). 35U. K. Laemmli, Nature (London) 227, 680 (1979). 36M. Montal, R. Anholt, and P. Labarca, in "Iron Channel Reconsfitution" (C. Miller, ed.), p. 157. Plenum, New York, 1986. 37M. G. McNamee, O. T. Jones, and T. M. Fon& in "Ion Channel Reconstitution" (C. Miller, ed.), p. 231. Plenum, New York, 1986. 3a Avanti Polar Lipids (Birmingham, AL). 39Laboratory Supplies Company, Inc. (HicksviUe,NY).
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about 1 nmol of toxin binding sites, is sedimented and suspended in 620/~1 of 0.5% lipid in 2CNP100(pH 8.0), made by diluting the above 3.5% lipid solution with 2CNP 100 (pH 8.0). All steps are at 4 °. After being stirred for 1 hr, the suspension is centrifuged 30 min in a microcentrifuge. The supernatant is mixed with an equal volume of the 3.5% lipid solution and agitated for 30 min. This mixture is dialyzed successively against 500 ml of NP100 (pH 8.0) for 14 to 16 hr, against 500 ml of 180 mMsucrose, 10 m M Tris-C1, pH 8.0, 0.02% NaN 3 for 10 to 12 hr, and against a fresh 500 ml of the same sucrose solution for about 18 hr. To assay ligand-gated channel activity in purified AChR, AChR containing about 1 nmol of toxin binding sites in the cholate-asolectin mixture (see above) is made up to a final concentration of 2% lipid in 2CNP100 (pH 8.0) with the 3.5% lipid mixture and 2CNP100 (pH 8.0) and dialyzed as above. Prior to reconstitution, ion-exchange resin for the flux assay is prepared as follows. One hundred grams of AG 50W-X8 (100-200 mesh) ~s in the hydrogen form is washed twice with 500 ml of methanol and twice with 1 liter of deionized water. The resin is equilibrated with 200 ml of 1 M Tris base by stirring gently for 30 min and is washed 3 times with 1 liter of deionized water. The resin is similarly equilibrated with 200 ml of 1 M HC1 and washed with water. It is reequilibrated with 200 ml of 1 M Tris base, allowed to settle, suspended in 1 liter of water, and allowed to settle. The equilibration with Tris is repeated until the supernatant pH is 8.0. Sodium azide is added to 0.02%, and the resin is stored at 4 °. Prior to the flux assay, 1.4 ml of resin is added to disposable columns and is washed first with 2 ml of 1 mg/ml bovine serum albumin, 0.02% NaN 3 and second with 2 ml of 180 m M sucrose, l0 m M Tris-C1 (pH 8.0), 0.02% NaN 3. The latter solution is used below as the eluant. In the flux assay, 22Na is diluted with the above eluant to contain about 145,000 counts/min (cpm) per 10 #1. Ten microliters of this 22Na and 10/zl of either 700 pA//carbamylcholine in eluant or eluant alone are added to the bottom of a tube. Fifty microliters of reconstituted AChR is rapidly added and mixed with an Eppendorf pipette. With a second pipette, the mixture is immediately transferred to the top of the column of resin. This is followed immediately with 1.5 ml ofeluant. The entire output from the column, approximately 1.5 ml, contains the vesicles and the included 22Na but not free 22Na. This is collected in a 7-ml scintillation vial; 5 ml of Scintisol is added, and the vial is counted. Although a white gel forms, more Scintisol does not result in higher efficiency counting. Under the above conditions, approximately 4 times as many counts (about 600 cpm) are taken up by the reconstituted vesicles in the presence of carbamylcholine than in its absence (about 150 cpm).
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bilayers by the septum-supported, vesicle-derived bilayer technique, which has been reviewed by Schindler elsewhere in this series.37 In the planar bilayer configuration, the ion conductance activity of purified calcium channels is modulated by calcium channel activators and inhibitors as expected. Activation of the channel by Bay K8644 required phosphorylation by cAMP-dependent protein kinase, and the level of channel activity was greatly increased by phosphorylation as observed in ion flux experiments. However, in the planar bilayer system, individual ion conductance events ranged in size from 0.9 to 60 pS, in contrast to the 20-pS values observed with patch pipette methods. Nevertheless, the results of ion flux, patch pipette, and macroscopic bilayer methods together show that purified skeletal muscle calcium channels retain selective ion conductance activity that is modulated appropriately by calcium channel-modulating drugs and protein phosphorylation. 37 H. Schindler, this series, Vol 171, p. 225.
[36] P u r i f i c a t i o n a n d R e c o n s t i t u t i o n o f N i c o t i n i c Acetylcholine Receptor
By AMITABHCHAK and ARTHUR KARLIN Introduction The nicotinic acetylcholine receptor (AChR) was the first receptor, and certainly the first ligand-gated channel, to be purified (see Ref. 1 for review). This success during 1973 and 1974 was due largely to the availability of very rich sources, namely, the electric tissues of electric rays (family Torpedinidae) and the electric eel (Electrophorus electricus), and to the availability of reversible and irreversible ligands for the acetylcholine binding sites. Also, at that time there was increasing awareness of the properties of amphipathic, integral membrane proteins, of the hydrophobic effect, and of detergents.2 It became clear that the techniques used to purify water-soluble proteins could be applied to membrane proteins in detergent complexes. The minimal criteria for the choice of detergent were that the protein was efficiently solubilized and that at least one assayable property, binding in the case of receptors, was preserved. For receptors like the AChR that are ligand-gated channels, although 1A. Karlin, in "The Cell Surface and Neuronal Function" (C. W. Cotman, G. Poste, and G. L. Nicolson, eds.), p. 191. Elsevier/North-Holland Biomedical, Amsterdam, 1980. 2 C. Tanford, "The Hydrophobic Effect," 2nd Ed. Wiley, New York, 1980.
METHODS IN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPrem,Inc. Allrightsofreproductlonin any form re,fred,
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binding is preserved in detergent solution, the critical functional properties of gating and ion conduction are observed only in membranes. Therefore to assay these functions in purified ligand-gated channels, one must reincorporate them into artificial membranes. Also, studies of receptor topology and interactions with other membrane proteins and lipids require either native membranes or reconstituted membranes. We describe the preparation, starting with Torpedo californica electric tissue, of AChR-rich native membranes, solubilized and purified AChR, and AChR reconstituted in liposomes. Acetycholine receptor subtypes have also been purified from other tissues (e.g., muscle and neuronal tissue) and from many other species (see Ref. 3 for review). Recently, many subtypes have been characterized genetically rather than biochemically (see Ref. 4 for review). Electric Tissue Live rays5 are dissected, and the tissue is stored in liquid nitrogen. Alternatively, tissue can be purchased already frozen. In the former case, working in the cold room and wearing rubber gloves, we remove the ray from its container and immediately pith it. The two electric organs fill the "wings." A scalpel is used to cut around the edges of the kidney-shaped organs. The dermal layer on the dorsal side is peeled off with a surgical damp, following which the organ is shelled offthe ventral surface by blunt dissection. The electric organs are weighed, placed on an enameled metal tray, and cut into approximately 2 cm chunks, which are dropped one by one into liquid nitrogen for quick freezing. We have stored tissue under liquid nitrogen for several months. A medium-sized ray yields 600 to 1200 g of electric tissue. Preparation of M e m b r a n e s Rich in Nicotinic Acetylcholine R e c e p t o r For the preparation of AChR-rich membranes, ~ approximately 120 g of frozen Torpedo tissue is thawed in 300 ml of PE buffer.7 Subsequent 3j. Lindstrom,R. Schoepfer,and P. Whiting,Mol. Neurobiol. l, 281 (1987). 4T. Claudio, in "Frontiers of Neurobiology: Molecular Neurobiology"(D. Glover and D. Haines,eds.),p. 63. IRL, London, 1989. 5PacificBiomarine(Venice,CA) or Marinus(LongBeach,CA). 6C. Czajkowski,M. DiPaola, M. Bodkin,G. Salazar-Jimenez,E. Holtzman,and A. Karlin, Arch. Biochem. Biophys. 272, 412 (1989). 7Abbreviationsfor solutions: PE bufferis l0 mM sodiumphosphate, 1 mM EDTA, pH 7; NPxxx is xxx mM NaCl, l0 mM sodiumphosphate, 1 mM EDTA, 0.02% NAN3,pH 7.0, unless otherwisespecifiedin parentheses,as, for example,NP100 (pH 8.0); TNPxxxis 0.2% Triton X-100 in NPxxx; 2CNPxxx(pH 7.4) is 2% sodiumcholatein NPxxx(pH 7.4); 0.5 CNPxxx(pH 7.4) is 0.5%sodiumcholatein NPxxx(pH 7.4); B bufferis 0.2%TritonX-100, 10 mM NaC1, l0 mM MOPS, pH 7.4.
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operations are carried out at 4 ° . The mixture containing nearly thawed tissue is placed in a food processor 8 and minced by four bursts of 30 sec each. Most of the foam is allowed to settle. One-third of the suspension at a time is homogenized at full speed, twice, for 1 min, in a 500-ml flask of a homogenizer9 fitted with two sharpened blades. The resulting suspension (H1) is centrifuged in two JA-14 rotor bottles 1° at 6000 rpm for 10 min. The supernatants are pooled and saved on ice. The pellets are suspended in I00 ml of PE buffer and rehomogenized in a 250-ml flask,9 at full speed, twice, for 1 min. This supernatant is added to the saved pool, which is then filtered through eight layers of cheesecloth. The pellets (PI) are discarded. The supernatant (S1) is centrifuged in 26-ml polycarbonate bottles in a 60 Ti rotor 1° at 45,000 rpm for 40 min. The resulting supernatant ($2) is discarded. The pellets (P2) are suspended in 100 ml of 40% (w/v) sucrose in PE buffer and homogenized in a 250-ml flask at full speed twice for 1 min. This homogenate (H2) is centrifuged in polycarbonate bottles in a 60 Ti rotor at 60,000 rpm for 140 min. The resulting supernatants ($3) are discarded. The pellets (P3) are suspended in 40 ml PE buffer and homogenized in a 100-ml flask, 9 with one blade on the shaft, at full speed for l min. Homogenized P3 is frozen and stored in liquid nitrogen in 4-ml aliquots. Discontinuous sucrose density gradients with 12 ml of 44% (w/v) sucrose in PE buffer under l0 ml of 40% (w/v) sucrose are made in 60 Ti rotor polycarbonate bottles. P3 is removed from liquid nitrogen and thawed. Eighteen milliliters of P3 is mixed with 3.6 ml of 3 M NaCI in PE buffer to give a final concentration of 0.5 M NaC1. This suspension is passed in and out l0 times through a blunted 22-gauge, 3.5-inch spinal needle attached to a 30-ml plastic syringe. A 3-ml aliquot of this suspension (H3) is layered over each of six discontinuous gradients, and the gradients are centrifuged in a 60 Ti rotor at 60,000 rpm for 140 min. After centrifugation, two bands and a pellet are visible. The second band at the interface of the 40 and 44% sucrose layers is collected by suction from the top. These second bands are pooled and diluted with 4 to 5 volumes of PE buffer to a volume of 150 ml and centrifuged at 60,000 rpm in a 60 Ti rotor for 80 min. The resulting pellets in 4 ml of PE buffer are passed in and out, 10 times, through a 22-gauge needle, and this suspended AChRrich membrane is stored in liquid nitrogen. The yield is 1 to 2 mg of protein at a speofic activity of about 3 nmol of a-bungarotoxin binding sites per milligram of protein. The protein of this membrane therefore is about 40% AChR. Further purification can be s Cuisinart Model DLC-10 Plus. 9 Homogenizer Model Virtis-45, The Virtis Co., Inc. (Garcliner, NY). ~0 Rotors and centrifuges from Beckman Instruments Inc. (Palo Alto, CA).
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obtained by extraction o f peripheral m e m b r a n e proteins with dilute N a O H ( p H 1 1) ~t or with lithium diiodosalicylate, ~2but with consequent increased disordering o f the membrane. Also, affinity partitioning has given high specific activities.~3 Purification of Detergent-Solubilized Receptor The detergent used to solubilize the AChR should give a good yield while maintaining structural and functional integrity. Triton X-100 efficiently solubilizes the A C h R and maintains its snake curarimimefic toxinbinding activity and, with some alteration, its binding o f acetylcholine (ACh) and other small ligands.~ Ligand-gated channel activity o f the AChR cannot be recovered once it has been solubilized in Triton X-100, even though the solubilized A C h R can be reincorporated into liposomes) 4 For reconstitution of channel activity, solubilization and purification in cholate and phospholipid mixtures are required) 5 For structural studies, the higher yield and purity obtained by purification in Triton X-100 is preferred. We describe solubilization and purification o f AChR both in Triton X-100 and in a cholate and phospholipid mixture.
Preparation of Affinity Gel Bromoacetylcholine Bromide (BAC) ~6. T o 18.4 g o f choline bromide is added dropwise, over 40 rain, with stirring, 24.2 g o f b r o m o a c e t y l bromide. The viscous mixture is stirred for an additional 75 rain in an ice bath. T o the mixture is slowly added 75 ml o f absolute ethanol. The white crystalline product is recrystallized twice from 400 ml o f 2-propanol. The yield is 67% and the product melts at 135 o _ 136 °. BAC is stored desiccated at 4 ° and is not opened for weighing until at r o o m temperature. Affinity geP 7. Affi-Gel 10, ~s 25 ml in isopropanol as supplied, is washed 1~R. R. Neubig, E. K. Krodel, N. D. Boyd, and J. B. Cohen, Proc. Nat. Acad. S¢i. U.S.A. 76, 690 (1979). ~2j. Elliot, S. G. Blanehard, W. Wu, J. Miller, C. D. Strader, P. Hartig, H.-P. Moore, J. Rats, and M. A. Rat~ery,Biochem. J. 185, 667 (1980). t3 G. Johansson, R. Gysin, and S. D. Flanagan, a*.Biol. Chem. 256, 9126 (1981). ~4M. G. MeNamee, C. L. Weill, and A. Karfin, in "Protein-Lip,and Interactions" (H. Sund and G. Blauer, eds.), p. 316. deGruyter, Berlin, 1975. ~5M. Epstein and E. Racker, J. Biol. Chem. 253, 6660 (1978). ~6V. N. Damle, M. MeLaughlin, and A. Karlin, Biochem. Biophys. Res. Commun. 84, 845 (1978). t7 We previously used Afli-Gel401, TM a crosslinked agarose gel with attached homocysteinyl residues at about 10/tmol per ml packed gel, which we reacted with BAC to form the cholinocarboxymethyl adduct [A. Reynolds and A. Karlin, Biochem. 17, 2035 (1978)]. Bio-Rad recently discontinued manufacture of Atii-Gel 401. We now use Afli-Gel 10, a N-hydroxysuccinimideester linked to crosslinked agarose by a neutral 10 atom spacer. is Bio-Rad Laboratories, Inc. (Richmond, CA).
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PURIHCATION OF ION CHANNEL PROTEINS AND GENES
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in a Buchner funnel with 250 ml of cold water. The gel should not be allowed to dry during this or any subsequent step. The wet gel is transferred to a beaker with 75 ml 13.3 mMcystamine in 100 m M HEPES (pH 8.0). This slurry is shaken for 1 hr at room temperature. The gel is transferred to a Buchner and washed with 50 ml HEPES buffer and 4 × 50 ml water. The gel is mixed in a beaker with 50 ml 20 m M dithiothreitol in 200 m M TRIS buffer (pH 8.0) and shaken for 1 hr. The gel is washed with 600 ml water. Aliquots (100/zl) of the gel and of the supernatant are saved for sulfhydryl assay. The gel is mixed with 50 ml 20 m M BAC in 200 m M NaC1, 50 m M sodium phosphate buffer (pH 7.0) and shaken for 1 hr. The gel is washed with 500 ml water. Before the reaction with BAC, the gel contains about 10/zmol sulfhydryl per ml gel, and after BAC it contains no free sulfhydryls. The affinity gel is made before the membrane extract. The gel can be stored at 4 ° in 0.02% NaN 3 for a few days. Assayfor Sulfhydryl Groups in Gel. A gel sample is pipetted into a tared 12-ml conical glass centrifuge tube and sedimented for 1 min. The supernatant is carefully removed with a pulled out Pasteur pipette, and the packed gel is weighed. The gel is suspended by vortexing in 1 ml of 1 m M 5,5'-dithiobis(2-nitrobenzoic acid) in 200 m M Tris-C1 (pH 8.0) and after 1 min is sedimented. To 100/zl of the supernatant is added 1 ml of 200 m M Tris-C1 (pH 8.0), and the absorbance at 412 nm of this mixture is determined. A control without gel is treated similarly. A molar extinction coefficient of 13,600 is used to determine the sulfhydryl concentration in the cuvette, from which the sulfhydryl titer of the gel can be calculated. If after treatment with BAC the gel contains unreacted sulfhydryl groups, these can be blocked with N-ethylmaleimide.
Crude Membrane Preparation For the preparation of crude membranes, 19 600 g of Torpedo tissue is partially thawed and added to 2400 ml of 1 m M EDTA, pH 7.4. (If preservation of Torpedo AChR dimers is desired, 2 m M N-ethylmaleimide is added here to block free sulfhydryls, preventing the reduction of the disulfide linking the monomers in dimer.) All subsequent steps are carried out at about 4*. Two milliliters of 0.5 M phenylmethylsulfonyl fluoride (PMSF) in ethanol is added, and the tissue is homogenized for 60 sec in a 1-gallon commercial blender a° set at "low." This homogenate is filtered through four layers of cheesecloth and centrifuged in about 10 JA-14 polycarbonate bottles at 14,000 rpm for 90 rain. The supernatant is discarded. The pellets are collected in 1200 ml of the EDTA buffer and 19 Co L. WeiU, M. G. McNamee, and A. Karlin, Biochem. Biophys. Res. Commun. 61, 997 (1974). 20 Waring Products Division (New Hartford, CT).
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homogenized in the blender set at "high" for 20 see. This homogenate is centrifuged in the JA-14 rotor at 14,000 rpm for 90 rain. The supernatant is removed carefully from the loose pellets and discarded. The pellets are pooled in a 250-ml graduated cylinder and brought up to a volume of 173 ml with EDTA buffer; 21 ml of 500 m M NaC1, 100 m M sodium phosphate, l0 m M EDTA, 0.2% sodium azide, pH 7, is added. This suspension is homogenized in the l-liter container for the blender set at "medium" for 20 sec.
Extraction of Membrane in Triton X-I O0 For Triton X-100 extraction, 19 the crude membrane preparation is stirred vigorously in a 250-ml Erlenmeyer flask, while 50/21 of 0.5 M P M S F in ethanol and 16 ml of 40% Triton X-100 in water is added. The final medium contains 3% Triton X-100, 50 m M NaCI, 10 m M sodium phosphate, 1 m M EDTA, 0.02% azide pH 7. After being stirred for 1 hr at 4 °, the mixture is transferred to eight 60 Ti rotor tubes and centrifuged at 60,000 rpm for 30 rain. The extract is carefully removed from the pellets and pooled. It can be stored overnight in ice and in the refrigerator, or it can be loaded slowly onto the affinity column, for elution the next day.
Affinity Chromatography in Triton X-IO0 The extract can be adsorbed onto the affinity gel either prior to pouting the column or after pouring the column. '7,19 All operations are carried out in a cold room. In the first case, 24 ml of affinity gel is washed 3 times with 25 ml of TNP50. 7 The affinity gel and extract are stirred together with a magnetic bar at slow speed in a 250-ml beaker for 2 hr. The suspension is transferred to four 50-ml centrifuge tubes and sedimented for 4 rain. The supernatant is carefully removed. The gel is washed twice with TNP50. About 25 ml of TNP50 is added to the gel, and the slurry is transferred with a broken-off Pasteur pipette into a 1.5-era diameter glass column, using more TNP50 to complete the transfer as needed. During this process, eluate is collected at 1 drop]see at a drop size of 15 #1. In the second ease, the affinity gel is poured into a column in TNP50. The extract is loaded onto this column overnight at a flow rate of 10 to 15 ml/hr, controlled by a peristaltic pump. After the extract is loaded, the column is washed with 200 ml of TNP50 at 60 ml/min. After loading the extract by either method, the column is washed with 200 ml of TNP100 at 60 ml/hr. Finally, the AChR is eluted with 50 ml of 10 m M earbamylcholine in TNP90 at 30 ml/hr. This eluate is collected in 5-ml fractions, and the AChR elutes in fractions 2 through 7. These fractions in 1-ml aliquots are frozen in liquid nitrogen for assay and use. A
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typical yield is about 30 mg of AChR with specific activities in the different fractions ranging from 5 to 8 nmol curarimimetic toxin binding sites per milligram of protein. (The theoretical maximum specific activity is 7 nmol sites/mg, but there are inaccuracies in both the binding site and protein assays.) The above procedure results in pure or nearly pure AChR from Torpedo californica. In variations of the above method, slight additional purification and, in addition, resolution of monomeric and dimeric forms of AChR were obtained by sucrose density gradient centrifugation? TM In purifying AChR from less concentrated sources than Torpedo, other laboratories have used, in addition to affinity chromatography, chromatography on DEAE-cellulose, 22 on immobilized lectin, 23 and on immobilized antibody. ~ Also, affinity chromatography has been carried out on agarose linked either to other small ligands 21 or to various elapid snake toxins. 25
Purification in Cholate and Phospholipid Mixture The following variations of the above procedures preserve the ligandgated channel function of the AChR, which one can assay on reincorporation of the AChR into membrane. 26 A crude membrane fraction is prepared as above, up to the last sedimentation, except typically at one-sixth the scale, starting with 100 g of tissue. The l-liter blender container is used in place of the 1-gallon container. The crude membrane pellet is suspended in a final volume of 60 ml of 0.5% asolectin 27 in 2CNP100 (pH 7.4) 7 by homogenization at "low" for 20 sec. The mixture is stirred for 1 hr and then centrifuged at 60,000 rpm for 30 min. The supernatant is used immediately or stored in ice overnight. The BAC-Affi-Gel 10 affinity gel described above is washed with 0.0625% asolectin in 0.5CNP50 (pH 7.4), and 6 ml is poured into a 0.9-cm-diameter glass column. The supernatant is pumped onto the column at 30 ml/hr. The column is washed at the same rate successively with 25 ml of 0.5CNP50 (pH 7.4) and 50 ml of 0.5CNP100 (pH 7.4), and the AChR is eluted with 15 ml of 10 m M carbamylcholine in 0.5CNP90 (pH 7.4). The elution of protein can be followed in this case by its absorbance at 280 nm. Carbamylcholine-eluted fractions containing appreciable protein are stored in liquid nitrogen. 21 A. Karlin, M. G. McNamee, C. L. Weill, and R. Valderrama, in "Methods in Receptor Research" (M. BLecher, ed.), p. 1. Dekker, New York, 1976. 22 G. Biesecker, Biochemistry 12, 4403 (1973). 23 S. Froehner, C. G. Reiness, and Z. Hall, J. Biol. Chem. 252, 8589 (1977). u p. j. Whiting and J. Lindstrom, Biochemistry 25, 2082 (1986). 25 j. Lindstrom, B. Einarson, and S. Tzartos, this series, Vol. 74, p. 452. 26 R. L. Huganir, M. A. ScheU, and E. Racker, FEBSLett. 108, 155 (1979). 27 Associated Concentrates (Woodside, NY).
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Assays Acetylcholine Binding Sites Elapid snake venom contains basic polypeptide toxins which bind with high specificity and affinity to the ACh binding sites. Radioactive derivatives of these toxins are available. For example, the widely used a-bungarotoxin (Bgtx) is commercially available as an ~25I derivative,28 and Naja naja siamensis toxin 3 is readily tritiated. 29 In the former case, the iodinated toxin (initially about 140 Ci/mmol) is diluted 50 to 100 times with unlabeled ~-bungarotoxin 3° prior to use. 3~ We henceforth refer to the high-specific activity ~25I-labeledbungarotoxin diluted with unlabeled Bgtx simply as [125I]Bgtx. On the day of use, [~2SI]Bgtx is diluted to about 140 pmol/ml in 100 m M NaC1, 10 m M MOPS, pH 7.4, 0.2% Triton X-100, and 0.1 mg/ml bovine serum albumin. Two 25-mm DE-81 filters32 are placed on each screen of a filtration manifold and are washed with 4 ml of buffer B. Fifty microliters of diluted [~25I]Bgtxis added to 50/tl of AChR containing about 2 pmol of sites and mixed well. After 30 rnin at room temperature, the mixture is diluted with 5 ml of cold B buffer7 and poured onto the doubled DE-81 filters. 29,33 Mild suction at 50 to 100 m m Hg pressure difference is applied, the tube is washed onto the filter with another 5 ml of B buffer, and the filter is washed with 15 ml more of B buffer, now with a pressure difference of 200 m m Hg for faster filtration. After the filters are placed in the bottom of a scintillation vial, 250/tl of 1 M acetic acid is pipetted onto 2s New England Nuclear Corp. (Boston, MA). 29 V. N. Damle and A. Karlin, Biochemistry 17, 2039 (1978). 3o Sigma Chemical Co. (St. Louis, MO). 31 One should determine that [~2sI]Bgtx and Bgtx bind equivalently to AChR as follows. A fixed quantity of AChR is titrated with the diluted [~2~I]Bgtx.If uniabeled Bgtx bound with higher affinity than {125I]Bgtx,then the quantity of 12sIbound would reach a maximum at equivalence, when all Bgtx plus [125I]Bgtx were bound and all toxin binding rites were occupied, and then the 125Ibound would dc~'rea~ with increasing total Bgtx concentration to a new steady level as the excess unlabeled Bgtx competed with the [1251]BgUtfor the toxin binding sites; that is, there would be a cusp in the binding curve around equivalence. If no cusp is observed, one may conclude that labeled and unlabeled Bgtx bind equivalently for the purposes of tbis assay. In addition, one should routinely determine the fraction of t25Iin the [12SI]Bgtx preparation that is bound by excess AChR. This fraction is typically about 85%. One determines the factor (cpm/mol) for converting radioactivity to the quantity of Bgtx by counting a known quantity of [t25I]Bgtx plus Bgtx on filters, by multiplying these counts by the fraction of |2sI that could be bound by AChR, and by dividing by the quantity of Bgtx plus [1251]Bgtxin the sample. 32 Whatman, Inc. (Clifton, N J). 33 R. P. Klett, B. W. Fulpius, D. Cooper, M. Smith, E. Reich, and L. D. Possam, J. Biol. Chem. 252, 4811 (1973).
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the filters. After I 0 to 20 min at room temperature, 5 ml of Scintisol~ or equivalent counting cocktail is added, and the vial is placed in a scintillation counter.
Composition by Sodium Dodecyl Sulfate Gel Electrophoresis Standard Laemmli a5 conditions are used with a 7.5% acrylamide resolving gel. The sample is prepared initially by dissolving in 2% sodium dodecyl sulfate (SDS), 10 m M Tris, pH 8.0, 10 m M DTT, and holding at 50 ° for 30 min. (Higher temperatures can lead to degradation ofsubunits.) Thirty millimolar N-ethylmaleimide is added, and after an additional 15 min 9 volumes of acetone is added. After a few minutes, the precipitate is sedimented in a clinical centrifuge and washed with 5 volumes of acetone. The pellet is dried in a vacuum desiccator and dissolved in Laemmli sample buffer. Reduced and alkylated AChR yields four bands (a, fl, 7, and 6) with apparent masses of about 40, 48, 58, and 64 kDa? 9 If DTT and 2-mercapto-ethanol in the Laemmli sample buffer are omitted, the 64-kDa band (6) is diminished, and a 130-kDa band (~ dimer) is seen instead. Purity is indicated by the absence of other bands on the gel.
Reconstitution and Flux A s s a y 15,36,37 For reconstitution and flux assays,~mt,37 ligand-gated channel activity is more effectively reconstituted in a mixture of phosphatidylethanolamine (PE), 3s phosphatidylserine (PS), 3s and cholesterol than in asolectin. A P E P S - cholesterol mixture, 6: 3: 1 by weight, is made at 100 mg/ml in chloroform. Two hundred microliters of this mixture is dried to a film with a stream of N2 and then dried further under reduced pressure for 3 hr. To the dried lipid is added 570/zl of 2CNPI00 (pH 8.0). 7 The final concentration of lipid is 3.5%. The mixture is vortexed and sonicated under N2 in a bath sonicator 39 until the mixture is opalescent (about 5 min). Native Torpedo membrane vesicles are quite leaky. The ligand-gated channel activity of the AChR can be more reproducibly assayed after solubilization and reconstitution into liposomes. Membrane, containing 34Isolab, Inc. (Akron, OH). 35U. K. Laemmli, Nature (London) 227, 680 (1979). 36M. Montal, R. Anholt, and P. Labarca, in "Iron Channel Reconsfitution" (C. Miller, ed.), p. 157. Plenum, New York, 1986. 37M. G. McNamee, O. T. Jones, and T. M. Fon& in "Ion Channel Reconstitution" (C. Miller, ed.), p. 231. Plenum, New York, 1986. 3a Avanti Polar Lipids (Birmingham, AL). 39Laboratory Supplies Company, Inc. (HicksviUe,NY).
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about 1 nmol of toxin binding sites, is sedimented and suspended in 620/~1 of 0.5% lipid in 2CNP100(pH 8.0), made by diluting the above 3.5% lipid solution with 2CNP 100 (pH 8.0). All steps are at 4 °. After being stirred for 1 hr, the suspension is centrifuged 30 min in a microcentrifuge. The supernatant is mixed with an equal volume of the 3.5% lipid solution and agitated for 30 min. This mixture is dialyzed successively against 500 ml of NP100 (pH 8.0) for 14 to 16 hr, against 500 ml of 180 mMsucrose, 10 m M Tris-C1, pH 8.0, 0.02% NaN 3 for 10 to 12 hr, and against a fresh 500 ml of the same sucrose solution for about 18 hr. To assay ligand-gated channel activity in purified AChR, AChR containing about 1 nmol of toxin binding sites in the cholate-asolectin mixture (see above) is made up to a final concentration of 2% lipid in 2CNP100 (pH 8.0) with the 3.5% lipid mixture and 2CNP100 (pH 8.0) and dialyzed as above. Prior to reconstitution, ion-exchange resin for the flux assay is prepared as follows. One hundred grams of AG 50W-X8 (100-200 mesh) ~s in the hydrogen form is washed twice with 500 ml of methanol and twice with 1 liter of deionized water. The resin is equilibrated with 200 ml of 1 M Tris base by stirring gently for 30 min and is washed 3 times with 1 liter of deionized water. The resin is similarly equilibrated with 200 ml of 1 M HC1 and washed with water. It is reequilibrated with 200 ml of 1 M Tris base, allowed to settle, suspended in 1 liter of water, and allowed to settle. The equilibration with Tris is repeated until the supernatant pH is 8.0. Sodium azide is added to 0.02%, and the resin is stored at 4 °. Prior to the flux assay, 1.4 ml of resin is added to disposable columns and is washed first with 2 ml of 1 mg/ml bovine serum albumin, 0.02% NaN 3 and second with 2 ml of 180 m M sucrose, l0 m M Tris-C1 (pH 8.0), 0.02% NaN 3. The latter solution is used below as the eluant. In the flux assay, 22Na is diluted with the above eluant to contain about 145,000 counts/min (cpm) per 10 #1. Ten microliters of this 22Na and 10/zl of either 700 pA//carbamylcholine in eluant or eluant alone are added to the bottom of a tube. Fifty microliters of reconstituted AChR is rapidly added and mixed with an Eppendorf pipette. With a second pipette, the mixture is immediately transferred to the top of the column of resin. This is followed immediately with 1.5 ml ofeluant. The entire output from the column, approximately 1.5 ml, contains the vesicles and the included 22Na but not free 22Na. This is collected in a 7-ml scintillation vial; 5 ml of Scintisol is added, and the vial is counted. Although a white gel forms, more Scintisol does not result in higher efficiency counting. Under the above conditions, approximately 4 times as many counts (about 600 cpm) are taken up by the reconstituted vesicles in the presence of carbamylcholine than in its absence (about 150 cpm).
