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.     

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.     

Binding properties of agonists and antagonists to distinct allosteric states of the nicotinic acetylcholine receptor are incompatible with a concerted model

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.     

Stopped flow kinetics of carbamylcholine binding to membrane bound acetylcholine receptor

Conformational changes upon binding of carbamylcholine to acetylcholine receptor-enriched membrane fragments have been observed by stopped-flow methods using the fluorescent probe ethidium bromide. A model consistent with both equilibrium and kinetic experiments is proposed in which the receptor binds two molecules of carbamylcholine with high affinity in a non-cooperative manner followed by binding of a third and possibly a fourth molecule with increasingly lower affinity. The receptor ligand precomplexes isomerize to different non-interconvertible complexes depending on the number of ligands bound. This kinetic model fits the data for carbamylcholine interactions with receptor prepared initially either in a low or high affinity form for ligands.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.     

Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding

The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

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.     

A fluorescence probe of acetylcholine receptor conformation and local anesthetic binding

The fluorescent dye ethidium bromide binds to the acetylcholine receptor with an apparent K_d of $\text{3 μ}\text{M}$ and a stoichiometry of 1 molecule of ethidium per α-bungarotoxin site. Time dependent fluorescent increases were observed upon addition of carbamylcholine, the amplitude and half-time of which were dependent on the Carb¹ concentration. It appeared that these fluorescence increases resulted from a lowering of the K_d for ethidium as the AcChR-Carb complex underwent an isomerization from low to high affinity form(s) for carb, and more ethidium was bound. Titration with the local anesthetic procaine led to ethidium fluorescence increases at low procaine concentrations, followed by a fluorescence decrease at higher procaine concentrations to that level induced by saturating α-bungarotoxin. Thus it appeared that the ethidium binding site either interacted with or was identical with local anesthetic binding site(s).     

Identification of nicotinic acetylcholine receptor amino acids photolabeled by the volatile anesthetic halothane

To identify inhalational anesthetic binding domains in a ligand-gated ion channel, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with [(14)C]halothane and determined by Edman degradation some of the photolabeled amino acids in nAChR subunit fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography. Irradiation at 254 nm for 60 s in the presence of 1 mM [(14)C]halothane resulted in incorporation of approximately 0.5 mol of (14)C/mol of subunit, with photolabeling distributed within the nAChR extracellular and transmembrane domains, primarily at tyrosines. GammaTyr-111 in ACh binding site segment E was labeled, while alphaTyr-93 in segment A was not. Within the transmembrane domain, alphaTyr-213 within alphaM1 and deltaTyr-228 within deltaM1 were photolabeled, while no labeled amino acids were identified within the deltaM2 ion channel domain. Although the efficiency of photolabeling at the subunit level was unaffected by agonist, competitive antagonist, or isoflurane, state-dependent photolabeling was seen in a delta subunit fragment beginning at deltaPhe-206. Labeling of deltaTyr-212 in the extracellular domain was inhibited >90% by d-tubocurarine, whereas addition of either carbamylcholine or isoflurane had no effect. Within M1, the level of photolabeling of deltaTyr-228 with [(14)C]halothane was increased by carbamylcholine (90%) or d-tubocurarine (50%), but it was inhibited by isoflurane (40%). Within the structure of the nAChR transmembrane domain, deltaTyr-228 projects into an extracellular, water accessible pocket formed by amino acids from the deltaM1-deltaM3 alpha-helices. Halothane photolabeling of deltaTyr-228 provides initial evidence that halothane and isoflurane bind within this pocket with occupancy or access increased in the nAChR desensitized state compared to the closed channel state. Halothane binding at this site may contribute to the functional inhibition of nAChRs.     

Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes

Two conformational states of the nicotinic acetylcholine receptor have been investigated by cryoelectron microscopy of flattened vesicular crystals grown from Torpedo marmorata postsynaptic membranes. One was obtained from the vesicles without acetylcholine present, and is presumed to correspond to the native, or resting state; the other was obtained from the vesicles after exposure to 100 microM to 5 mM carbamylcholine (an acetylcholine analogue) and is presumed to correspond to a desensitized state. Both conformations were determined in three-dimensions to a resolution of 18 A, sufficient to reveal the configurations of the five subunits around the central ion channel over most of their length. The subunits of either structure have a similar appearance, consistent with their amino acid homology. They are each aligned almost parallel to the axis of the receptor, conferring a high degree of pentagonal symmetry to the bilayer portion and a contiguous region on the synaptic side. Their external surfaces form a pronounced ridge in the bilayer portion, which broadens toward the synaptic end. Comparison of features in the two three-dimensional maps reveals that carbamylcholine induces a quaternary rearrangement, involving predominantly the delta-subunit. The densities corresponding to this subunit are tilted by approximately 10 degrees tangential to the axis of the receptor over a large fraction of its length, and become misaligned relative to the densities corresponding to the other four subunits. The gamma-subunit is also affected, being displaced slightly away from the axis of the receptor. The alpha- and beta-subunits may be affected on a more localized scale. The overall changes are most pronounced in the synaptic region, where the ligand-binding site is located, and in the cytoplasmic region, which may be closer to the gate of the channel. The physiological process of desensitization appears to be associated with a structural transition in which the subunits switch to a less symmetrical configuration.     

Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.     

A conformational intermediate between the resting and desensitized states of the nicotinic acetylcholine receptor

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.     

The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.     

Dissecting the chemistry of nicotinic receptor-ligand interactions with infrared difference spectroscopy.

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.     

Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.     

Identification of the sites of incorporation of [3H]ethidium diazide within the Torpedo nicotinic acetylcholine receptor ion channel

The binding sites of ethidium, a noncompetitive antagonist of the nicotinic acetylcholine receptor (nAChR), have been localized in the Torpedo nAChR in the desensitized state by use of a photoactivatible derivative, [(3)H]ethidium diazide. At 10 microM [(3)H]ethidium diazide, incorporation into the alpha-, beta-, and delta-subunits was inhibited by the presence of phencyclidine (PCP). Within the alpha-subunit, the incorporation was mapped to a 20-kDa fragment beginning at alphaSer-173 and containing the first three transmembrane segments, alphaM1, alphaM2, and alphaM3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing alphaM1 and one containing both alphaM2 and alphaM3. Within alphaM2, specific incorporation was present in alphaLeu-251 and alphaSer-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the delta-subunit with S. aureus V8 protease generated a 14-kDa and a 20-kDa fragment, both of which began at Ile-192 and contained PCP-inhibitable labeling. The 14-kDa fragment, containing deltaM1 and deltaM2, was further digested to generate a 3-kDa fragment, containing deltaM2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained deltaM1, deltaM2, and deltaM3, generated two fragments with incorporation, one containing the deltaM1 segment and the other containing deltaM2 and deltaM3. These results establish that in the desensitized state of the nAChR, the high-affinity binding site of ethidium is within the lumen of the ion channel and that the bound drug is in contact with amino acids from both the M1 and M2 hydrophobic segments.     

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.     

3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure

Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.     

Potential state-selective hydrogen bond formation can modulate activation and desensitization of the α7 nicotinic acetylcholine receptor

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.