Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

Phosphorylation of phosphatidylinositol associated with the nicotinic acetylcholine receptor of Torpedo californica

When isolated, detergent solubilized and affinity chromatographically purified nicotinic acetylcholine receptor of Torpedo californica electric organ is incubated with [gamma-32P]ATP/Mg2+, phosphatidylinositol 4-phosphate (PIP) is formed from receptor associated phosphatidylinositol (PI). This receptor associated endogenous kinase activity is enhanced by orthovanadate and, remarkably, also by acetylcholine. Exogenously added PI-kinase only increases the phosphorylation rate if vanadate is present. PIP as the main phosphorylation product (up to 95%) remains bound to the beta-, gamma- and delta-subunits of the receptor and to the receptor associated v-protein. The alpha-subunits do not carry 32p phosphate; no phosphatidylinositol 4,5-bisphosphate formation has been observed. Concomitant to lipid phosphorylation tyrosine and serine residues are phosphorylated (5% of total incorporated 32P phosphate).     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.     

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [³H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 μM. These classes of sites exist in approximately equal numbers and all [³H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.     

Interactions of charatoxins and nereistoxin with the nicotinic acetylcholine receptors of insect cns and Torpedo electric organ

Interactions of charatoxin (4-methylthio-1,2-dithiolane; ChTX) and four openchain analogs as well as nereistoxin (NTX) with acetylcholine (ACh) receptors were studied using biochemical assays on the Torpedo electric organ and honey bee brain receptors and using electrophysiological assays on the response of the cell body of the fast coxal depressor motoneuron (D_f) of the cockroach Periplaneta americana to ACh. The actions of ChTXs were complex. Except for ChTX Xl, they all potentiated the ACh-induced current in Periplaneta neurons, but at higher concentrations all ChTXs, except for ChTX XII, caused voltage-dependent block of this current. All CHTXs inhibited binding of [³H]perhydrohistrionicotoxin in the presence of ACh to the highaffinity noncompetitive blocker site on the Torpedo receptor, but all, except for ChTX XI, potentiated its binding in absence of ACh. The actions of ChTXs on the honey bee brain receptor were quite different from those on the Torpedo receptor. They inhibited, or had no effect on, [¹²⁵I]α-bungarotoxin (α-BGT) binding to the Torpedo receptor, but all ChTXs, except for ChTX I, potentiated its binding to the honey bee receptor. It is suggested that the action of ChTXs on nicotinic ACh-receptors resulted from binding to lowaffinity noncompetitive blocker site. On the other hand, NTX was more potent than ChTXs on nicotinic ACh-receptors, and some similarities were noted between the actions of NTX on Torpedo and honey bee receptors NTX had a weak agonistlike effect in both cases and possibly bound to the ACh binding sites as well as the high-affinity noncompetitive blocker site. Thus the mechanisms of action of ChTXs and NTX on nicotinic ACh-receptors are different, and there are also differences in the responses to these toxins between receptors of insect central nervous system and Torpedo electric organ.     

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.     

Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

A photoactivatable analogue of phosphatidylserine, 125I-labeled 4-azidosalicylic acid-phosphatidylserine (125I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin (a crude soybean lipid extract) vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the alpha subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR alpha subunit that incorporated 125I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the alpha subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the alpha subunit incorporated little or no detectable amount of probe.     

Mapping the lipid-exposed regions in the Torpedo californica nicotinic acetylcholine receptor.

To identify regions of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used 1-azidopyrene (1-AP) as a fluorescent, photoactivatable hydrophobic probe. For AchR-rich membranes equilibrated with 1-AP, irradiation at 365 nm resulted in covalent incorporation in all four AchR subunits with each of the subunits incorporating approximately equal amounts of label. To identify the regions of the AchR subunits that incorporated 1-AP, subunits were digested with Staphylococcus aureus V8 protease and trypsin, and the resulting fragments were separated by SDS-PAGE followed by reverse-phase high-performance liquid chromatography. N-terminal sequence analysis identified the hydrophobic segments M1, M3, and M4 within each subunit as containing the sites of labeling. The labeling pattern of 1-AP in the alpha-subunit was compared with that of another hydrophobic photoactivatable probe, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID). The nonspecific component of [125I]TID labeling [White, B., Howard, S., Cohen, S. G., & Cohen, J.B. (1991) J. Biol. Chem. 266, 21595-21607] was restricted to the same regions as those labeled by 1-AP. The [125I]TID residues labeled in the hydrophobic segment M4 were identified as Cys-412, Met-415, Cys-418, Thr-422, and Val-425. The periodicity and distribution of labeled residues establish that the M4 region is alpha-helical in nature and indicate that M4 presents a broad face to membrane lipid.     

Photolabeling the Torpedo nicotinic acetylcholine receptor with 4-azido-2,3,5,6-tetrafluorobenzoylcholine, a partial agonist

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.     

Interactions between alpha-conotoxin MI and the Torpedo marmorata receptor alpha-delta interface

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.     

Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor

Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.     

Get effective polyclonal antisera in one month

According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund‘s complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund‘s incomplete adjuvant) were followed every 2-4 weeks. In general,high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4months. This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis.It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection. You could gain the high titer of the antisera right after the first sample B injection in one month. This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells.     

Benzophenone-type photoactivatable derivatives of alpha-neurotoxins and alpha-conotoxins in studies on Torpedo nicotinic acetylcholine receptor

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom alpha-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions, derived from arylazide or diazirine photolabels, that alpha-neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.     

Electrostatic determinants of the ion channel control of the nicotinic acetylcholine receptor of Torpedo californica

The patch clamp K⁺-conductance G of the nicotinic acetylcholine receptor (AcChoR) dimer (M_r≈ 590 000) of Torpedo californica, reconstituted in lipid vesicles, which decreases with increasing Ca²⁺-concentration in the range 0.1≤[Ca²⁺]/mM≤2, can be quantitatively rationalized by Ca²⁺-binding to negatively charged sites, causing charge reversal reducing the normal K⁺-accumulation in the channel vestibules. Cleavage of the sialic acid residues (up to 20±2 per dimer) reduces the K⁺-accumulation factor α = G₀/G_∞ from α = 3±0.8 of the normal AcChoR to α = 2±0.7 for the desialyated AcChoR. Desialysation also decreases the Ca²⁺-sensitivity of the conductance from G₀ = 96.6±6 pS at [Ca²⁺]→0 of the normal AcChoR to G₀ = 84.2±6 pS. Endogenous hyperphosphorylation (to up to 28±4 phosphates per dimer) enhances the vestibular K⁺-accumulation to α = 3.6±0.7, without affecting the Ca²⁺-dissociation equilibrium constant K_Ca = 0.34± 0.05 mM at 295 K (22 °C). Most interestingly, even in the absence of AcCho, the hyperphosphorylated AcChoR dimer exhibits spontaneously long-lasting open channel events (τ = 200±50 ms). At [AcCho] = 2 μM there are two open states (τ ₁ = 20±10 ms, τ ₂ = 140±60 ms) whereas the normal AcChoR dimer has only one open state (τ = 6±4 ms). – Physiologically important is that (i) the sialic acid and phosphate residues render the AcChoR conductance sensitive to control by divalent ions and (ii) the channel behavior of the hyperphosphorylated AcChoR without AcCho appears to indicate pathophysiologically high phosphorylation activity of the cell leading, among others, to myasthenic syndromes. Received: 10 November 1997 / Revised version: 12 January 1998 / Accepted: 7 March 1998     

Expression of the four subunits of the Torpedo californica nicotinic acetylcholine receptor in Saccharomyces cerevisiae

Yeast expression vectors were constructed containing complementary DNA encoding the alpha-, beta-, gamma-, and delta-subunits of the Torpedo californica nicotinic acetylcholine receptor under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter. All four plasmids were integrated into the yeast genome of a single yeast cell. The resulting yeast strain synthesized polypeptides novel to yeast that had the molecular weights and antigenic properties similar to the authentic T. californica receptor alpha-, gamma, and delta-subunits. The beta-subunit polypeptide could not be detected in this yeast strain, even though the poly(A)+ RNA from this strain contained all the information necessary for the expression of functional acetylcholine receptors in Xenopus laevis oocytes. The replacement of the beta-subunit mRNA 5'-untranslated leader and its N-terminal signal sequence by the corresponding alpha-subunit sequences, however, resulted in the expression of the beta-subunit polypeptide in yeast grown at 5 degrees C.     

Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica.

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242] to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor. The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by high-resolution proton NMR. Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N-acetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal alpha-galactose residue(s) linked to C3 of beta-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Gal beta 1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.     

Interactions between tachykinins and diverse,human nicotinic acetylcholine receptor subtypes

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP) on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotinestimulated function measured using⁸⁶Rb⁺ efflux assays of human ganglionic (α3β4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC₅₀∼2.3 μM) or of human muscle-type (α1β1γδ) nAChR expressed in TE671/RD clonal cells (IC₅₀∼21 μM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC₅₀∼2.1 μM). Neurokinin A and eledoisin inhibit function (extrapolated IC₅₀ values between 60 and 160 μM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR is as sensitive as PC12 cell α3β4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of nAChR function, SP fails to affect binding of [³H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation of nervous system activity.     

Interaction of the acetylcholine (nicotinic) receptor protein from Torpedo marmorata electric organ with monolayers of pure lipids

Membrane fragments rich in cholinergic (nicotinic) receptor protein were purified from the electric organ of Torpedo marmorata. Their lipid composition is essentially characterized by the prominence of cholesterol, phosphatidylethanolamine and phosphatidylcholine, long-chain fatty acyl constituents, and the absence of sphingomyelin. Solubilised receptor was purified from these fragments and the concentration of sodium cholate lowered by dialysis to 0.01% (w/v). When this preparation was injected under a lipid monolayer, an increase of surface pressure developed, which was not observed with the detergent alone nor in the absence of lipid film. When covalently radiolabelled receptor preparations were injected at a constant surface pressure the radioactivity recovered with the film was proportional to the increase in area. It is concluded that the pressure or area increases are due to the penetration of the cholinergic receptor protein into the lipid film. Incorporation experiments into films formed from various pure lipids showed that the protein interacts more readily with cholesterol than with ergosterol, phosphatidylcholine, or other phospholipids. Its affinity is also higher for long-chain phosphatidylcholines than for short-chain ones. The degree of unsaturation and fluidity of the 3-sn-phosphatidylcholine (lecithin) films are of secondary importance. Parallel experiments with covalently and non-covalently labelled receptor preparations showed that part of the protein recovered with the film lost its alpha-toxin binding ability during the penetration. Similar data were obtained with the receptor purified from Electrophorus electricus electric organ.