Probing the structure of the nicotinic acetylcholine receptor with 4-benzoylbenzoylcholine, a novel photoaffinity competitive antagonist

[(3)H]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K(D) = 1.4 microm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [(3)H]Bz(2)choline results in the covalent incorporation of [(3)H]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [(3)H]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gammaVal-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at deltaAsp-99. Amino-terminal sequence analysis identified the homologous residues gammaLeu-109 and deltaLeu-111 as the primary sites of [(3)H]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gammaLeu-109/deltaLeu-111.     

Endogenous and exogenous proteolysis of the acetylcholine receptor from torpedo californica

Purified acetylcholine receptor reconstituted into liposomes catalyzes carbamylcholine-dependent ion flux [10]. An endogenous protease activated by Ca²⁺ gives rise to an acrylamide gel pattern of the receptor with the 40,000-dalton subunit apparently as the major component. Exogenous proteases nick the proteins so extensively that the acrylamide gel pattern reveals polypeptides of 20,000 daltons or less. In either case the receptor sediments at 9S, indicating that the polypeptide chains associated. Moreover, the nicked receptors bind α-bungarotoxin and catalyze carbamylcholine-dependent ion flux after reconstitution.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane α-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 Å at its narrowest, to 8.6 Å at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

Probing conformational changes in the nicotinic acetylcholine receptor by Fourier transform infrared difference spectroscopy.

We have developed a Fourier transform infrared (FTIR) difference method for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR). Our approach is to deposit reconstituted nAChR membranes in a thin film on the surface of a germanium internal reflection element, acquire FTIR spectra in the presence of bulk aqueous solution using attenuated total reflection, and then trigger conformational changes by sequentially flowing a buffer either with or without an agonist past the film surface. Using the fluorescent probe, ethidium bromide, it is demonstrated that the method of nAChR film deposition does not affect the ability of the receptor to undergo the resting-to-desensitized state transition. The difference of FTIR spectra of nAChR films recorded in the presence and absence of agonists reveal highly reproducible infrared bands that are not observed in the difference of spectra recorded with only buffer flowing past the film surface. Some of the bands are assigned to changes in protein secondary structure and to changes in the structure of individual amino acid residues. Bands arising from the vibrations of the agonist bound to the receptor are also observed. The results demonstrate that FTIR difference spectroscopy can detect structural changes in the nAChR that occur upon the binding of ligands. The technique will be an effective method for investigating nAChR structure and function as well as receptor-drug interactions.     

Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.     

Evolution of nicotinic acetylcholine receptor subunits

A phylogenetic tree of a gene family of nicotinic acetylcholine receptorsubunits was constructed using 84 nucleotide sequences of receptor subunitsfrom 18 different species in order to elucidate the evolutionary origin ofreceptor subunits. The tree constructed showed that the common ancestor ofall subunits may have appeared first in the nervous system. Moreover, wesuggest that the alpha 1 subunits in the muscle system originated from thecommon ancestor of alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, and beta 3in the nervous system, whereas the beta 1, gamma, delta, and epsilonsubunits in the muscle system shared a common ancestor with the beta 2 andbeta 4 subunits in the nervous system. Using the ratio (f) of the number ofnonsynonymous substitutions to that of synonymous substitutions, wepredicted the functional importance of subunits. We found that the alpha 1and alpha 7 subunits had the lowest f values in the muscle and nervoussystems, respectively, indicating that very strong functional constraintswork on these subunits. This is consistent with the fact that the alpha 1subunit has sites binding to the ligand, and the alpha 7-containingreceptor regulates the release of the transmitter. Moreover, the windowanalysis of the f values showed that strong functional constraints work onthe so-called M2 region in all five types of muscle subunits. Thus, thewindow analysis of the f values is useful for evaluating the degree offunctional constraints in not only the entire gene region, but also thewithin-gene subregion.     

Molecular investigations on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a well-understood member of the ligand-gated ion channels superfamily. The members of this signaling proteins group, including 5HT₃, GABA_A, glycine, and ionotropic glutamate receptors, are thought to share common secondary, tertiary, and quaternary structures on the basis of a very high degree of sequence similarity. Despite the absence of X-ray crystallographic data, considerable progress on structural analysis of nAChR was achieved from biochemical, mutational, and electron microscopy data allowing the emergence of a three-dimensional image. Photoaffinity labeling and site-directed mutagenesis gave information on the tertiary structure with respect to the agonist/antagonist binding sites, the ion channel, and its selectivity filter. nAChR is an allosterical protein that undergoes interconversion among several conformational states. Time-resolved photolabeling was used in an attempt to elucidate the structural changes that occur in nAChR on neurotransmitter activation. Tertiary and quaternary rearrangements were found in the cholinergic binding pocket and in the channel lumen, but the structural determinant and the functional link between the binding of agonist and the channel gating remain unknown. Time-resolved photolabeling of the functional activated A state using photosensitive agonists might help in understanding the dynamic process leading to the interconversion of the different states.     

Computed pore potentials of the nicotinic acetylcholine receptor

Electrostatic surface potentials in the vestibule of the nicotinic acetylcholine receptor (nAChR) were computed from structural models using the University of Houston Brownian Dynamics program to determine their effect on ion conduction and ionic selectivity. To further determine whether computed potentials accurately reflect the electrostatic environment of the channel, the potentials were used to predict the rate constants for diffusion-enhanced fluorescence energy transfer; the calculated energy transfer rates are directly comparable with those determined experimentally (see companion article by Meltzer et al. in this issue). To include any effects on the local potentials by the bound acceptor fluorophore crystal violet, its binding site was first localized within the pore by fluorescence energy transfer measurements from dansyl-C6-choline bound to the agonist sites and also by simulations of binding using Autodock. To compare the computed potentials with those determined experimentally, we used the predicted energy transfer rates from Tb3+ chelates of varying charge to calculate an expected potential using the Boltzmann relationship. This expected potential (from -20 to -40 mV) overestimates the values determined experimentally (from -10 to -25 mV) by two- to fourfold at similar conditions of ionic strength. Although the results indicate a basic discrepancy between experimental and computed surface potentials, both methods demonstrate that the vestibular potential has a relatively small effect on conduction and selectivity.     

Diversity in primary structure and function of neuronal nicotinic acetylcholine receptor channels.

Neuronal nicotinic acetylcholine receptors are oligomeric protein complexes whose component subunits are each encoded by a family of homologous genes. The current challenge is to determine the functional contributions of the related subunits to the receptor-linked ion channels they compose and to uncover the physiological impact of the distinct channel classes expressed in vivo. In the past year, new approaches to the analysis of these receptors have yielded important insights into their stoichiometry, pharmacology and functional properties.     

Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila

Acetylcholine is a major excitatory neurotransmitter in the central nervous system of insects. Using DNA probes of the Torpedo nicotinic acetylcholine receptor (AChR) we have isolated two overlapping cDNA clones encoding a putative neuronal AChR protein from the fruitfly, Drosophila melanogaster. The predicted mature protein consists of 497 amino acids, has a calculated mol. wt of 57 340 and shows extensive homology to known AChR subunits from different species along its entire amino acid sequence. Northern analysis revealed a hybridizing mRNA of 3.2 kb in late embryo and in pupae. Expression of the corresponding AChR gene thus characterizes periods of neuronal differentiation in Drosophila.     

Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosophila

[This corrects the article on p. 1503 in vol. 5.].     

Interaction of gephyrotoxin and indolizidine alkaloids with the nicotinic acetylcholine receptorion channel complex of torpedo electroplax

The interactions of eighteen natural and synthetic gephyrotoxin and indolizidine alkaloids with binding sites on nicotinic acetylcholine receptor channel (AChR) complex fromTorpedo californica electric organ were investigated using two radiolabeled probes, [³H]perhydrohistrionicotoxin and [³H]phencyclidine. Both gephyrotoxins and indolizidines were moderately active inhibitors of the binding of these probes (K _i's=0.1–20 M), but did not interact with the actylcholine binding site. Structure-activity relationships indicate an important contribution of hydrophobic interactions to both gephyrotoxin and indolizidine binding. The stereoconfiguration of the alkaloids had little effect on binding. Carbamylcholine enhanced the affinity of certain alkaloids up to 6 to 8-fold suggesting that interactions with open or desensitized conformations of the AChR complex are favored over interactions with resting conformations.     

Angiogenesis and the role of the endothelial nicotinic acetylcholine receptor

An endothelial nicotinic acetycholine receptor (nAChR) mediates endothelial proliferation, survival, migration and tube formation in vitro, and angiogenesis in vivo. Exogenous nicotine stimulates this angiogenic pathway. This action of nicotine may contribute to tumor angiogenesis and tumor growth; atherosclerotic plaque neovascularization and progression; and other tobacco-related diseases. The endothelial nAChR mediates an angiogenic pathway that is interdependent with growth factor mediated pathways, as shown by pharmacological and molecular studies. The characterization of this new angiogenic pathway may provide a new therapeutic avenue for disorders of insufficient or pathological angiogenesis.