Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

Subunit-selective contribution to channel gating of the M4 domain of the nicotinic receptor

The muscle nicotinic receptor (AChR) is a pentamer of four different subunits, each of which contains four transmembrane domains (M1-M4). We recently showed that channel opening and closing rates of the AChR depend on a hydrogen bond involving a threonine at position 14' of the M4 domain in the alpha-subunit. To determine whether residues in equivalent positions in non-alpha-subunits contribute to channel gating, we mutated deltaT14', betaT14', and epsilonS14' and evaluated changes in the kinetics of acetylcholine-activated currents. The mutation epsilonS14'A profoundly slows the rate of channel closing, an effect opposite to that produced by mutation of alphaT14'. Unlike mutations of alphaT14', epsilonS14'A does not affect the rate of channel opening. Mutations in deltaT14' and betaT14' do not affect channel opening or closing kinetics, showing that conserved residues are not functionally equivalent in all subunits. Whereas alphaT14'A and epsilonS14'A subunits contribute additively to the closing rate, they contribute nonadditively to the opening rate. Substitution of residues preserving the hydrogen bonding ability at position 14' produce nearly normal gating kinetics. Thus, we identify subunit-specific contributions to channel gating of equivalent residues in M4 and elucidate the underlying mechanistic and structural bases.     

Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state

The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two α subunits are rotated relative to its non-α subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the α subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the αG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the α subunits.     

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an alpha(2)betaepsilondelta composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15' of the M1 domain is phenylalanine in alpha subunits while it is isoleucine in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant alpha subunit (alphaF15'I) as well as those containing the reverse mutations in the non-alpha subunits (betaI15'F, deltaI15'F, and epsilonI15'F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the beta subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of betaI15'F of AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant in the diliganded receptor, and a significant increase opening in the absence of agonist. Mutations at betaI15' showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an approximately 70% closed-state-like environment for the beta15' position at the transition state of gating. The overall results identify position 15' as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.     

Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor

Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltage-clamped oocytes. For all the substitutions the concentration for half-maximal activation, EC ₅₀, is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2- to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC ₅₀s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs. Received: 29 January 2001/Revised: 14 May 2001     

Mechanics of channel gating of the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer β-sheet composed of β9 and β10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O ↔ C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.     

Interfacial properties of the M1 segment of the nicotinic acetylcholine receptor

We have studied the thermodynamic, surface, and structural properties of alphaM1 transmembrane sequence of the nicotinic acetylcholine receptor (nAChR) by using Langmuir monolayer, FT-IR spectroscopy and molecular dynamics simulation techniques in membrane-mimicking environments. M1 spontaneously incorporates into a lipid-free air-water interface, showing a favourable adsorption free energy of -7.2 kcal/mol. A cross-sectional molecular area of 210 A(2)/molecule, a surface potential of 4.2 fV/molecule and a high stability of the film were deducted from pure M1 monolayers. FT-IR experiments and molecular dynamics simulations in membrane-mimicking environments (sodium-dodecyl-sulfate and CCl(4), respectively) indicate coexistence between helical and non-helical structures. Furthermore, mixed peptide-lipid monolayers and monolayer penetration experiments were performed in order to study the peptide-lipid interaction. Mixed with condensed lipids (dipalmitoyl-phosphocholine, and dipalmitoyl-phosphoglycerol), M1 shows immiscible/miscible behaviour at low/high peptide concentration, respectively. Conversely, a complete miscible peptide-lipid interface is observed with liquid-expanded lipids (palmitoyl-oleoyl-phosphocholine, and palmitoyl-oleoyl-phosphoglycerol). Peptide penetration experiments demonstrate that the M1 peptide preferentially interacts with zwitterionic phosphocholine interfaces.     

Tryptophan substitutions reveal the role of nicotinic acetylcholine receptor alpha-TM3 domain in channel gating: differences between Torpedo and muscle-type AChR

A recent tryptophan scanning of the alpha-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A., Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here we extend the study of the alpha-TM3 domain to the muscle-type AChR by examining functional consequences of single tryptophan substitutions at five conserved positions (alphaM282, alphaF284, alphaV285, alphaA287, and alphaI290) homologous to the alpha-TM3 positions that were recently characterized in the Torpedo AChR. Similarly to the Torpedo AChR, mutations alphaM282W and alphaV285W, which are presumed to face the interior of the protein, did not exhibit functional channel activity. Nevertheless, significant expression levels of these mutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR, tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalized macroscopic response. Single-channel recordings at low ACh concentration revealed that the increase in AChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration. These results suggest that tryptophan substitution directly affects channel gating, primarily the channel closing rate. Our results suggest that residues facing the interior of the protein (i.e., alphaM282 and alphaV285) may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations (i.e., F284W and I290W) presumably facing the lipid environment display a very different functional response between these two AChR species.     

Conformational dynamics of the alphaM3 transmembrane helix during acetylcholine receptor channel gating

Muscle acetylcholine receptors are synaptic ion channels that "gate" between closed- and open-channel conformations. We used Phi-value analysis to probe the transition state of the diliganded gating reaction with regard to residues in the M3, membrane-spanning helix of the muscle acetylcholine receptor alpha-subunit. Phi (a fraction between 1 and 0) parameterizes the extent to which a mutation changes the opening versus the closing rate constant and, for a linear reaction mechanism, the higher the Phi-value, the "earlier" the gating motion. In the upper half of alphaM3 the gating motions of all five tested residues were temporally correlated (Phi approximately 0.30) and serve to link structural changes occurring at the middle of the M2, pore-lining helix with those occurring at the interface of the extracellular and transmembrane domains. alphaM3 belongs to a complex and diverse set of synchronously moving parts that change structure relatively late in the channel-opening process. The propagation of the gating Brownian conformational cascade has a complex spatial distribution in the transmembrane domain.     

Insights from modelling the 3D structure of the extracellular domain of alpha7 nicotinic acetylcholine receptor

Based on the crystal structure of acetylcholine-binding protein, the three-dimensional structures of the extracellular domain, or the ligand-binding domains, of the monomer, homodimer, and homopentamer of the alpha7 nicotinic acetylcholine receptor were derived. The interface between two subunits, where the ligand-binding site is located, was investigated. Furthermore, an explicit definition of the ligand-binding pocket was illustrated that might provide useful clues for conducting various mutagenesis studies for finding drugs against schizophrenia and Alzheimer's disease.     

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor

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Multidimensional on-line screening for ligands to the alpha3beta4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase

The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.     

Antidepressants noncompetitively inhibit nicotinic acetylcholine receptor function

Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.     

Site-directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

The aim of this study was to present a new concept of site-directed reduction of disulfide bonds based upon the use of an affinity ligand harbouring a readily oxidizable dithiol. The cysteine bond involved in the acetylcholine binding site of the AChoR was specifically reduced by a carbamylcholine analogue. The ligand, in its oxidized form, was characterized by an affinity constant of 20 μM for the agonist binding site. In its dithiol form, it specifically reduced the disulfide between Cys-192 and Cys-193 on the -subunits of the nicotinic acetylcholine receptor. This reduction needed 10 times lower concentration when carried out with site-directed reducing agent (ARA) than with DTT, and was highly specific for the -subunits. The contribution of the carbamylcholine moiety of the site-directed reducing agent was clearly demonstrated in kinetic studies where reduction abilities of ARA, DTT and the methylated analogue of ARA (MeRA) were compared. At the same concentration (20 μM), DTT and MeRA had a 25 times lower initial rate of reduction than ARA. With 200 μM of DTT this initial reduction was still 4 times lower. Furthermore, the use of a maleimido undecagold cluster which specifically labeled the reduced nicotinic receptor opens the way to structural analysis of the agonist binding site by electron microscopy. These results demonstrate the potency of this kind of site-directed reducing agent for structural study of receptors or enzymes involving a disulfide bond in their active site.     

Insect nicotinic acetylcholine receptor: conserved neonicotinoid specificity of [(3)H]imidacloprid binding site

The insect nicotinic acetylcholine receptor (nAChR) is a major target for insecticide action. The rapidly expanding use of neonicotinoid insecticides of varied structures makes it increasingly important to define similarities and differences in their action, particularly for the first-generation chloropyridinyl compounds versus the second-generation chlorothiazolyl derivatives. We have shown with Musca domestica that a convenient and relevant determination of the neonicotinoid insecticide target is a binding site assay with [(3)H]imidacloprid ([(3)H]IMI). This study uses membranes from the aphids MYZUS: persicae and Aphis craccivora and from heads of the flies DROSOPHILA: melanogaster and Musca domestica to characterize the [(3)H]IMI binding sites relative to their number and possible species variation in structure-activity relationships. With emphasis on commercial neonicotinoids, six potent chloropyridinyl compounds are compared with the corresponding six chlorothiazolyl analogues (syntheses are given for chemicals prepared differently than previously described). The preference for chloropyridinyl versus chlorothiazolyl is not dependent on the insect species examined but instead on other structural features of the molecule. The chlorothiazolyl substituent generally confers higher potency in the clothianidin and desmethylthiamethoxam series and the chloropyridinyl moiety in the imidacloprid, thiacloprid, acetamiprid, and nitenpyram series. Two chlorothiazolyl compounds compete directly with the chloropyridinyl [(3)H]IMI for the same binding sites in MYZUS: and DROSOPHILA: membranes. This study shows conserved neonicotinoid specificity of the [(3)H]IMI binding site in each of the four insect species examined.     

Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating

The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closed<==>open isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (phi) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. phi was constant throughout the length of the alpha subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two alpha subunits move synchronously. Between subunits, the sequence of M4 motions is alpha-epsilon-beta. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.     

Photoaffinity labeling of functional states of the nicotinic acetylcholine receptor

The nicotinic acetycholine receptor was subjected to photoaffinity labeling in different conformational and functional states. The photolabel used was the ion-channel blocker [³H]-TPMP⁺. A procedure is described for isolating labeled -polypeptide chains from the receptor complex by preparative SDS-polyacrylamide gel electrophoresis. The photolabel was localized in the primary structure of the -chain. The site of labeling was found to be identical when photoaffinity labeling was performed in the resting, desensitized, or antagonist state, respectively.     

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.