Langmuir 1992,8, 3116-3121
3116
Langmuir-Blodgett Studies and Atomic Force Microscope Images of Nicotinic Acetylcholine Receptor Films T. L. Fare,*JJC. A. Palmer,+C. G. Silvestre,?D. H. Cribbs,*D. C. Turner,t S. L. Brandow,tJland B. P. Gabert Naval Research Laboratory, Code 6090, Center for Biomolecular Science and Engineering, Washington, D.C. 20375-5000, Department of Chemistry, University of Florida, Gainseville, Florida 32611-2024, and Division of Natural Science and Mathematics, St. Mary’s College of Maryland, St. Mary’s City, Maryland 20686 Received May 18,1992. In Final Form: September 16,1992 Lipid membranecomposition,subpham ionic strength, and pH affect the functionalityof transmembrane proteins in reconstituted systems. We present the surfacepressure-area and surface potential-area curves for nicotinic acetylcholine receptor (nAChR)films and nAChR-lipid filmswith various lipid compositions and subphase preparations. Addition of unsaturated lipid and cholesterol caused the protein-lipid films to exhibit a more fluid phase under compressionthan the nAChR film itaelf. The choice of buffer, as well as pH, also affected film isotherms. Lipid-protein films were transferred onto silicon substrates and atomic force microscope (AFM) images were obtained. Images of the transferred protein and lipid films show the presence of protein aggregates and regular structure in the lipid layer. The sizes of the observed features are consistent with the molecular dimensions of the lipids and nAChR. Introduction Communication of a stimulus from one cell to ita neighboring cells is mediated by the release of an agonist. For nervenerve or nervemuscle synapses, the agonist (acetylcholine) from the presynaptic nerve cell binds to nicotinic acetylcholine receptors (nAChR) on the postsynaptic cell. Agonist binding to a receptor site causes an associated ion channel that spans the lipid bilayer of the cell membrane to open.’ The ion current through the channel results in a depolarization of the cell membrane potential, which allows the neighboring cells to respond to or pass along the stimulus. Study of these receptor-ion channel proteins has been facilitated by protein purification and reconstitution into lipid bilayers. Lipid bilayers containing the nAChR have been formed using either the Montal-Mueller2or micropipet patch te~hnique.~ In both approaches a lipid-protein film is spread a t the air-water interface prior to forming a bilayer. Protein reconstitution into lipid bilayers has been a particularly useful system for studying the effects of various parameters on protein functionality. For example, this method has been used to determine current-voltage characteri~tics,2~~ transient response of the channel to neurostimulants,s‘ and temperature effects on channel f u n ~ t i o n .Systematic ~ studies have been performed to determined the influence of lipid membrane composition on protein functionality? Three conditions are considered neceesary to support nAChFi functionality: (1)appropriate membrane fluidity; (2) the presence of negatively charged lipide; (3) high concentrations of cholesterol. Generally for such studies, the protein concentration in the film is quite low so that individual proteins can be monitored in + Naval Research Laboratory. t Present addreee: Ohmicron Corp.,376Pheasant Run,Newtown,
PA 18940. St. Mary’s College of Maryland. 1 University of Florida. (1)Kandel, E. R.;Siegelbaum,S. A.; Schwartz, J. H. In Principale of Neural Science, 3rd ed.;Kandel, E. R.,Schwartz, J. H., J m l , T. M., Ed.;
Elsevier Science Pubhhing Co.: New York, 1991, Chapten 9 and 10. (2) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3661. (3) Montal,M.;Anholt,R.;Labarca,P. In Ion ChannelReconetitution; Mier, C., Ed.; Plenum Prw: New York, 1986; Chapter 8. (4) Fong, T. M.; MacNamee, M. G.Biochemistry 1986,25,830.
a 1 pm patch pipet diameter. In its native state, however, the nAChR concentration can be quite high, especially at synaptic juncti0ns.l In this paper, we investigate the Langmuir-Blodgett properties of reconstituted nAChR-lipid films with relatively high concentrations of protein. Lipids that satisfy the nAChR functionality support criteria (described above) are spread with the protein f h . Physical characterization of high concentration protein-lipid films was carried out under conditions designed to ensure the functionality of the protein. We present isotherms of films spread both on a subphase used for electrophysiologicalrecordings and on a subphase more amenable to film transfer. Effects of subphase and lipid environment on the surface pressure and surface potential of the films are shown. Atomic force microscope (AFM)images of transferred nAChR-lipid films have been obtained that show aggregates of protein with feature dimensions appropriate to lipid and protein molecules. Experimental Section 1. Protein Purification and Film Preparation. Affiiity purified nAChR was prepared from the electricorgan of Torpeda californica.6 Reconstituted vesicles with a high protein to lipid ratio were produced to increasethe amount of receptor deposited i n t h e f i i . ThenAChRboundtotheaffinitycolumnwaswashed and eluted with low lipid buffer containing 0.1 mg/mL dioleoyl phosphatidylcholine (DOPC) and 0.6% cholate in McNamee’s buffer A. The nAChR was eluted from the column with low lipid buffer containing 10 mM carbamoylcholine and then the carbamoylcholine and cholate were removed by extensive dialysis. The protein concentration of the reconstituted nAChR ranged from 0.3 ot 0.4 mg/mL aa determined by the procedure of Bradford? wing bovine y-globulin aa the protein standard. The purified nAChR was evaluated by SDS-PAGE in 10% slab gele that were stained with coomassie blue’ and waa found to contain only the four expected polypeptide bands for nAChR. The ability of the purified nAChR to bind iodinated a-bungarotoxin was measured by the DEAE filter disk assay as described (5) Jones, 0. T.; Ernest, J. P.; MacNamee, M. G. In Biological Membranee: A Practical Approach; Findley, J. B. C., Evans, W. H., Ed., IRL Preee: Oxford, Washington DC, 1987; pp 139-178. (6) Bradford, M. M. Anal. Biochem. 1976, 72, 248. (7) Laemmli, U. K. Nature (London) 1970,227,680.
0743-7463/92/2408-3116$03.00/0@ 1992 American Chemical Society
Studies of nAChR Film
by Walker et al! The specificactivity for a-bungarotoxin binding was in the range of 2.0-3.5 nmol/mg of protein. Lipids native to the fmh (those most tightly bound to the protein) and DOPC from the column were retained by the protein during purification. Lipid composition of native protein membranes is approximately 50 mol % phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin, and phosphatidylinositol). Unsaturated fatty acids make up a large percentage of the lipid composition, with high levels (25 mol % ) of decosahexanoyl(226)acylchaine, mainly associated with the ethanolamine phosphatidies. Most of the remaining lipid fraction is unesterified cholesteroL8 Lipids were obtained from Avanti Polar Lipids and are listed below for each f i bused. Chloroform or chloroform-ethanol (ratio41)was used to dissolve the lipids. All fiis were formed on a NIMA L-D2-SS f i i balance equipped with a Wilhelmy plate balance for surface pressure measurement. Doubly-distilled, deionized water (Nanopure 11, Barnstead) was used for the subphase. T w o different types of buffers were used for f i i preparation: (1) 1mM CaC12,l mM NaHlPO, adjusted to pH 7.0 (Ca buffer); (2) 10 mM 34Nmorpholino)propanesulfonicacid (MOPS), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaC1, adjusted to pH 7.4 or 8.4 (MOPS buffer). The Ca buffer was chosen to optimize f i i transfer and the MOPS buffer was chosen based on an electrophysiological formulation.' both native-nAChFt protein f i i and protein-lipid films were formed at the air-water interface for transfer onto substrates. For the protein fiis, the native-dChR vesicles were spread on the buffered subphase. By reduction of the ionic strength of the subphase below that within the vesicles, the vesicles were lysed to form a protein f i i at the air-water interface. We shall refer to such films as native-nAChR f i i . Protein-lipid f i i were formed in a two-step process: fiist, lipid was spread at the airwater interface from the organic solvent-lipid solution, and second, after the organic solvent evaporated, the nAChFt vesicle preparation was spread on the same air-water interface. S u p plemental lipids included in the protein f i i were chosen based on their effect on protein function and their utility in transferring fiis onto substrates.'JOJ1 On the basis of the protein concentration and the amount of lipid spread, we estimate the lipidto-protein ratio of these f i h to be about 5Oo:l (see Discussion below). There may be loss of protein into the subphaee by the lysing technique; however, this method does ensure that the protein is kept in ita native lipid environment during preparation and f i i spreading. Films were compressed at rates of 50-100 cm2/min and the surface pressurearea characteristics were recorded by computer. The surface potential was measured using an ionizing electrode (americium-241source) with a Ag/AgCl counter electrode. The voltage was recorded as a function of film area with a Keithley 617 potentiometer. Due to lose of material in the subphase and variation in protein concentration from sample to sample, the area per molecule for a film could not be accurately obtained; however, reproducible isotherms were obtained for individual f i i and qualitative comparison can be made among fiis. Film compressionis quoted as a function of trough area for consistency. 2. Protein-Lipid Film Isotherms. Native-nAChR and DOPC-DPPA-Cbolesterol-nAChR on MOPS Subphase. Films of native-nAChFt, DOPC-DOPPA-cholesterol, and DOPCDPPA-choleaterol-nAChR (DPPA = dipalmitoylphosphatidic acid) were spread on a MOPS buffer subphase at pH 7.4 and 8.4. An isotherm of 50 pL of native protein spread on the pH 7.4 MOPS-buffered subphase is shown in curve 1, Figure la; the i iis not stable protein film is in a liquid-expanded phase. The f and collapsesshortly after compressionto 20 mN/m is completed. A mixture of DOPC-DPPA-cholesterol(50pL, mole ratio 3 6 1433, no protein) was spread at room temperature on a MOPS(8) Walker, J. W.; Lukas, R. J.; McNamee, M. G. Biochemistry 1981, 209, 2191.
(9) McNamee, M.; Jones, 0. T.; Fong, T. M. In Zon Channel Reconstitution;Miller, C., Ed.;Plenum Prese: New York, 1986, Chapter 10, p 239. (10) Fare, T. L. Langmuir 1990,6, 1172. (11) Fare, T. L.; Rusin, K. M.; Bey, P. P., Jr. Sem. Actuators 1991,3, 61.
Langmuir, Vol. 8, No. 12,1992 3117 70
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Figure 1. Surface pressure and surface potential curves of the native-nAChR f i bon the MOPS-buffersubphase are presented. The constituents of the fiis for the surface pressurearea curves are as follows: (a, top) for subphase at pH 7.4 (1)native protein, (2) DOPC-DPPA-cholesterol, (3) DOPC-DPPA-cholesterol + nAChR; (b, middle) for subphase at pH 8.4 (1)native protein, (2) DOPC-PA-cholesterol; (3) DOPC-DPPA-cholesterol + nAChR, (c, bottom) surface potential-area for subphaee at pH 8.4 (1) native-nAChR fii, (2) DOPC-DPPA-cholesterol, (3) DOPC-DPPA-cholesterol + nAChFt. The lipid mole ratio for DOPC-DPPA-cholesterol in the film is 36:1433. Twice as much protein is spread at pH 8.4 as at pH 7.4. buffered subphaae at pH 7.4. The film is in a liquid-expanded phase, shown in curve 2 of Figure la, although, the lipid f i i is not as expanded as the protein f i iitself. Once the native protein and lipid films were characterized, lipid-nAChFt f i i (50 p L of lipid, 50 pL of protein) were spread and isotherms exhibiting a more pronounced liquid-expanded phase were obtained, shown in curve 3 of Figure la. Comparing the isotherms in Figure 1, we find that the addition of lipids to the native protein f i i has caused the native protein film to become slightly more liquid expanded. In Figure lb, we show a set of isotherms (50 p L of lipid, 100 p L of native protein, and 50 pL of lipid-100 p L of protein) similar
Fare et al.
3118 Langmuir, Vol. 8, No. 12, 1992
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