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.     

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.     

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).     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

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.     

Monovalent ion effects on acetylcholine receptor from Torpedo californica

The effects of the five Group I monovalent ions, Li, Na, K, Rb, and Cs, on [³H]acetylcholine binding to Triton X-100 solubilized acetylcholine receptor from Torpedo californica electroplax were examined. Acetylcholine binding was not greatly affected by Li or Na, but was inhibited by the other ions in the order Cs > Rb > K. The inhibition by K appeared to occur by a mechanism identical to that for d-tubocurarine inhibition of acetylcholine binding.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

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.     

Effects of lipids and detergents on the conformation of the nicotinic acetylcholine receptor from Torpedo californica.

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to characterize the effects of lipids and detergents on acetylcholine receptor (AChR) conformation. Affinity purified AChR reconstituted into dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA), and cholesterol showed the same pattern of [125I]TID-labeling and demonstrated the same reduction in labeling of all four subunits upon desensitization by the agonist carbamylcholine, as partially purified AChR in native lipids. On the basis of the patterns of [125I]TID incorporation, reconstitution into DOPC/DOPA also appeared to stabilize the resting (functional) conformation of the AChR, while reconstitution in DOPC/cholesterol or DOPC alone largely desensitized the AChR. The effects of lipids on the functional state of the AChR was determined independently by measuring the ability of AChR reconstituted into different lipid combinations to undergo the change in affinity for agonist diagnostic of desensitization. The dramatic reduction in the apparent levels of [125I]TID associated with the subunits of the AChR observed upon agonist-induced desensitization was shown not to be due to a change in affinity for tightly bound lipid. Solubilization of affinity purified AChR reconstituted into DOPC/DOPA/cholesterol by the non-ionic detergents octyl glucoside, Triton X-100, and Tween 20 (final detergent concentration = 1%) was shown to produce the same pattern of [125I]TID-labeling as desensitization by agonist, while solubilization in 1% sodium cholate appeared to stabilize a conformation of the AChR more similar to the resting state.     

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.     

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     

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.     

Disulfide bond cross-linked dimer in acetylcholine receptor from Torpedo californica

Acetylcholine receptor from $\text{Torpedo californica}$ electric tissue occurs in membrane, and is purified, as a mixture of monomer and dimer. Dimer is cross-linked by disulfide bonds involving one of the four polypeptide components of receptor, namely the one of apparent molecular weight of 64,000.     

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.     

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.