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.     

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.     

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.     

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.     

Functional divergence analysis of vertebrate neuronal nicotinic acetylcholine receptor subunits

Nicotinic acetylcholine receptors (nAChRs) are pentamers formed by subunits from a large multigene family and are highly variable in kinetic, electrophysiological and pharmacological properties. Due to the essential roles of nAChRs in many physiological procedures and diversity in function, identifying the function-related sites specific to each subunit is not only necessary to understand the properties of the receptors but also useful to design potential therapeutic compounds that target these macromolecules for treating a series of central neuronal disorders. By conducting a detailed function divergence analysis on nine neuronal nAChR subunits from representative vertebrate species, we revealed the existence of significant functional variation between most subunit pairs. Specifically, 44 unique residues were identified for the α7 subunit, while another 22 residues that were likely responsible for the specific features of other subunits were detected. By mapping these sites onto the 3?D structure of the human α7 subunit, a structure-function relationship profile was revealed. Our results suggested that the functional divergence related sites clustered in the ligand binding domain, the β2–β3 linker close to the N-terminal α-helix, the intracellular linkers between transmembrane domains, and the “transition zone” may have experienced altered evolutionary rates. The former two regions may be potential binding sites for the α7* subtype-specific allosteric modulators, while the latter region is likely to be subtype-specific allosteric modulations of the heteropentameric descendants such as the α4β2* nAChRs.

Communicated by Ramaswamy H. Sarma     

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.     

Structural and functional studies of the nicotinic acetylcholine receptor by solid-state NMR

Over the last seven years, solid-state NMR has been widely employed to study structural and functional aspects of the nicotinic acetylcholine receptor. These studies have provided detailed structural information relating to both the ligand binding site and the transmembrane domain of the receptor. Studies of the ligand binding domain have elucidated the nature and the orientation of the pharmacophores responsible for the binding of the agonist acetylcholine within the agonist binding site. Analyses of small transmembrane fragments derived from the nicotinic acetylcholine receptor have also revealed the secondary structure and the orientation of these transmembrane domains. These experiments have expanded our understanding of the channels structural properties and are providing an insight into how they might be modulated by the surrounding lipid environment. In this article we review the advances in solid-state NMR applied to the nicotinic acetylcholine receptor and compare the results with recent electron diffraction and X-ray crystallographic studies.Presented at the Biophysical Society Meeting on Ion channels – from structure to disease held in May 2003, Rennes, France     

Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel

Previous studies have established the presence of overlapping binding sites for the noncompetitive antagonists (NCAs) amobarbital, tetracaine, and 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine ([(125)I]TID) within the ion channel of the Torpedo nicotinic acetylcholine receptor (AChR) in the resting state. These well-characterized NCAs and competitive radioligand binding and photolabeling experiments were employed to better characterize the interaction of the dissociative anesthetics ketamine and thienylcycloexylpiperidine (TCP) with the resting AChR. Our experiments yielded what appear to be conflicting results: (i) both ketamine and TCP potentiated [(125)I]TID photoincorporation into AChR subunits; and (ii) ketamine and TCP had very little effect on [(14)C]amobarbital binding. Nevertheless, (iii) both ketamine and TCP completely displaced [(3)H]tetracaine binding (K(i)s approximately 20.9 and 2.0 microM, respectively) by a mutually exclusive mechanism. To reconcile these results we propose that, in the resting ion channel, TCP and ketamine bind to a site that is spatially distinct from the TID and barbiturate locus, while tetracaine bridges both binding sites.     

N-haloacetylimino neonicotinoids: potency and molecular recognition at the insect nicotinic receptor

This structure-activity relationship study for neonicotinoids with an N-haloacetylimino pharmacophore identifies several candidate compounds showing outstanding insecticidal potency and consequently leads to establishing their molecular recognition at an insect nicotinic receptor structural model, wherein the neonicotinoid halogen atoms (fluorine, chlorine, bromine, and iodine) variously interact with the receptor loops C-D interfacial niche via H-bonding and/or hydrophobic interactions.     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

Activation of skeletal muscle nicotinic acetylcholine receptors

Summary and Conclusions Work over the past ten years has greatly increased our understanding of both the structure and function of the muscle nicotinic acetylcholine receptor. There is a strongly supported general picture of how the receptor functions: agonist binds rapidly to sites of low affinity and channel opening occurs at a rate comparable to the agonist dissociation rate. Channel closing is slow, so the channel has a high probability of being open if both agonist-binding sites are occupied by ACh. Results of expression studies have shown that each subunit can influence AChR activation and have given a structural basis for the major physiological change known for muscle AChR, the developmental change in AChR activation. These general statements notwithstanding, there are still major areas of uncertainty which limit our understanding. We have emphasized these areas of uncertainty in this review, to indicate what needs to be done.First, the quantitative estimates of rate constants are not as strongly supported as they should be. The major reasons are twofold—uncertainties about the interpretation of components in the kinetic data and difficulties of resolving brief events. As a result, any inferences about the functional consequences of structural alterations must remain tenuous.Second, the functional behavior of individual AChRs is not as well understood as it should be. The kinetic behavior of an individual receptor clearly can be complex (section II). In addition, there is evidence that superimposed on this complexity there may be stable and kinetically distinguishable populations of receptors (section III). Until the basis for the kinetically defined populations is clarified, kinetic parameters for receptors of defined structure cannot be unambiguously obtained.Finally, it is not surprising that the studies of AChR of altered structure have not given definitive results. Two reasons should be apparent from the preceding points: there is not a fully supported approach for kinetic analysis, and the normal population may not be clearly defined. An additional complication is also emerging, in that the available data support the idea that specific residues distributed over all subunits may influence AChR activation. This possibility renders the task of analysis that much more difficult.The muscle nicotinic AChR has served as a prototype for the family of transmitter-gated membrane channels, which includes the muscle and neuronal nicotinic receptors, the GABA_A, the glycine and possibly the non-NMDA excitatory amino acid receptor (Stroud et al., 1990). It is interesting to note that the functional properties of the GABA_A receptor, probably the best-studied of the other members of the family are rather similar. In particular, opentime and burst durations show multiple components interpreted as reflecting openings of singly and doubly liganded receptors (Mathers & Wang, 1988; Macdonald et al., 1989), the distribution of gaps indicates a relatively complex gating scheme (Twyman et al., 1990; Weiss & Magleby, 1989), and multiple kinetic modes are likely to exist (Newland et al., 1991). The situation with regards to the effects of GABA_A receptor subunit stoichiometry is more complex than for muscle AChR (e.g., Luddens & Wisden, 1991), perhaps similar to that found for neuronal nicotinic AChR (Papke et al., 1989; Luetje et al., 1990; Luetje & Patrick, 1991). Overall, it appears that the unresolved questions about the muscle nicotinic AChR are not indications that this is an exceptionally complicated transmitter-gated channel. Rather, it appears to be a relatively straightforward member of the family, and the lessons we learn from studying it are likely to be directly applicable to other receptors.We thank many friends for discussion, including Tony Auerbach, Paul Brehm, Jim Dilger, Meyer Jackson, and Chuck Stevens who told us about data before publication. Research in the authors' laboratories is supported by grants from the NIH (CL and JHS) and the AHA (CL).     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Regulation of nicotinic acetylcholine receptor function by adenine nucleotides

1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.     

NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold

We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.     

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.     

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state NMR approach

Solid-state magic-angle spinning nuclear magnetic resonance (NMR) has sufficient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptor-toxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13C-,15N-labelled neurotoxin II. Despite the large size of the ligand-receptor complex ( > 290 kDa) and the high lipid content of the sample, we were able to detect and identify residues from the ligand. The comparison with solution NMR data of the free toxin indicates that its overall structure is very similar when bound to the receptor, but significant changes were observed for one isoleucine.     

Neuronal nicotinic acetylcholine receptors from Drosophila: two different types of alpha subunits coassemble within the same receptor complex

Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.