Purification and characterization of a nicotinic acetylcholine receptor from chick brain

Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.     

CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE‐INDUCED CURRENTS

Nicotinic acetylcholine receptors are ligand‐gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real‐time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine‐induced currents amplitudes. Moreover, co‐application with 0.5 μM α‐bungarotoxin completely inhibited nicotine currents whereas 10 μM d‐tubocurarine had a partial effect demonstrating that β1‐containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α‐bungarotoxin.     

Primary structure and expression of beta 2: a novel subunit of neuronal nicotinic acetylcholine receptors

A new subunit, beta 2, of the neuronal nicotinic receptor family has been identified. This subunit has the structural features of a non-agonist-binding subunit. We provide evidence that beta 2 can substitute for the muscle beta 1 subunit to form a functional nicotinic receptor in Xenopus oocytes. Expression studies performed in oocytes have demonstrated that three different neuronal nicotinic acetylcholine receptors can be formed by the pairwise injection of beta 2 mRNA and each of the neuronal alpha subunit mRNAs. The beta 2 gene is expressed in PC12 cells and in areas of the central nervous system where the alpha 2, alpha 3, and alpha 4 genes are expressed. These results lead us to propose that the nervous system expresses diverse forms of neuronal nicotinic acetylcholine receptors by combining beta 2 subunits with different agonist-binding alpha subunits.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Molecular studies of the neuronal nicotinic acetylcholine receptor family

Nicotinic acetylcholine receptors on neurons are part of a gene family that includes nicotinic acetylcholine receptors on skeletal muscles and neuronal alpha bungarotoxin-binding proteins that in many species, unlike receptors, do not have an acetylcholine-regulated cation channel. This gene superfamily of ligand-gated receptors also includes receptors for glycine and gamma-aminobutyric acid. Rapid progress on neuronal nicotinic receptors has recently been possible using monoclonal antibodies as probes for receptor proteins and cDNAs as probes for receptor genes. These studies are the primary focus of this review, although other aspects of these receptors are also considered. In birds and mammals, there are subtypes of neuronal nicotinic receptors. All of these receptors differ from nicotinic receptors of muscle pharmacologically (none bind alpha bungarotoxin, and some have very high affinity for nicotine), structurally (having only two types of subunits rather than four), and, in some cases, in functional role (some are located presynaptically). However, there are amino acid sequence homologies between the subunits of these receptors that suggest the location of important functional domains. Sequence homologies also suggest that the subunits of the proteins of this family all evolved from a common ancestral protein subunit. The ligand-gated ion channel characteristic of this superfamily is formed from multiple copies of homologous subunits. Conserved domains responsible for strong stereospecific association of the subunits are probably a fundamental organizing principle of the superfamily. Whereas the structure of muscle-type nicotinic receptors appears to have been established by the time of elasmobranchs and has evolved quite conservatively since then, the evolution of neuronal-type nicotinic receptors appears to be in more rapid flux. Certainly, the studies of these receptors are in rapid flux, with the availability of monoclonal antibody probes for localizing, purifying, and characterizing the proteins, and cDNA probes for determining sequences, localizing mRNAs, expressing functional receptors, and studying genetic regulation. The role of nicotinic receptors in neuromuscular transmission is well understood, but the role of nicotinic receptors in brain function is not. The current deluge of data using antibodies and cDNAs is beginning to come together nicely to describe the structure of these receptors. Soon, these techniques may combine with others to better reveal the functional roles of neuronal nicotinic receptors.     

Molecular biology of mammalian amino acid receptors

The amino acid receptor proteins are ubiquitous transducers of most excitatory and inhibitory synaptic transmission in the brain. In July 1987 two reports appeared describing the molecular cloning of a pair of subunits of the GABAA receptor (7) and one subunit of the glycine receptor (13). These papers sparked wide interest and led quickly to the concept of a ligand-gated receptor-ion channel superfamily that includes nicotinic acetylcholine receptors as well as certain amino acid receptors. The identification of additional subunits of each receptor followed; with the recent cloning of a kainate receptor subunit (14), only the NMDA receptor remains elusive. Several disciplines have been brought to bear on these receptor clones, including in situ hybridization and functional expression in Xenopus laevis oocytes and mammalian cell lines. In this review we compare cloning strategies that have been used for amino acid receptors and discuss structural similarities among the receptor subunits. Two findings that have arisen from molecular cloning and expression of these receptors receive special attention. First, the molecular heterogeneity of GABAA receptors is larger than expected from pharmacological studies of native receptors. Second, although the native receptors are thought to be heterooligomers, much like the model proposed for the nicotinic receptors, some individual amino acid receptor subunits can form functional receptor channels, presumably in a homomeric configuration. This review focuses, therefore, on what we have learned from cloning efforts about amino acid receptors and what might lie ahead in this field.     

Ubiquilin-1 regulates nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system

Nicotinic acetylcholine receptors found in the peripheral and central nervous system differ from those found at the neuromuscular junction. Recently we isolated a cDNA clone encoding the alpha subunit of a neuronal acetylcholine receptor expressed in both the peripheral and central nervous system. In this paper we report the isolation of a cDNA encoding the alpha subunit of a second acetylcholine receptor expressed in the central nervous system. Thus it is clear that there is a family of genes coding for proteins with sequence and structural homology to the alpha subunit of the muscle nicotinic acetylcholine receptor. Members of this gene family are expressed in different regions of the central nervous system and, presumably, code for subtypes of the nicotinic acetylcholine receptor.     

Dbeta3, an atypical nicotinic acetylcholine receptor subunit from Drosophila : molecular cloning, heterologous expression and coassembly

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.     

Thymopoietin,a thymic polypeptide,potently interacts at muscle and neuronal nicotinic α-bungarotoxin receptors

Current studies suggest that several distinct populations of nicotinic acetylcholine (ACh) receptors exist. One of these is the muscle-type nicotinic receptors with which neuromuscular nicotinic receptor ligands and the snake toxin alpha-bungarotoxin interact. alpha-Bungarotoxin potently binds to these nicotinic receptors and blocks their function, two characteristics that have made the alpha-toxin a very useful probe for the characterization of these sites. In neuronal tissues, several populations of nicotinic receptors have been identified which, although they share a nicotinic pharmacology, have unique characteristics. The alpha-bungarotoxin-insensitive neuronal nicotinic receptors, which may be involved in mediating neuronal excitability, bind nicotinic agonists with high affinity but do not interact with alpha-bungarotoxin. Subtypes of these alpha-toxin-insensitive receptors appear to exist, as evidenced by findings that some are inhibited by neuronal bungarotoxin whereas others are not. In addition to the alpha-bungarotoxin-insensitive sites, alpha-bungarotoxin-sensitive neuronal nicotinic receptors are also present in neuronal tissues. These latter receptors bind alpha-bungarotoxin with high affinity and nicotinic agonists with an affinity in the microM range. The function of the nicotinic alpha-bungarotoxin receptors are as yet uncertain. Thymopoietin, a polypeptide linked to immune function, appears to interact specifically with nicotinic receptor populations that bind alpha-bungarotoxin. Thus, in muscle tissue where alpha-bungarotoxin both binds to the receptor and blocks activity, thymopoietin also potently binds to the receptor and inhibits nicotinic receptors-mediated function. In neuronal tissues, thymopoietin interacts only with the nicotinic alpha-bungarotoxin site and not the alpha-bungarotoxin-insensitive neuronal nicotinic receptor population. These observations that thymopoietin potently and specifically interacts with nicotinic alpha-bungarotoxin-sensitive receptors in neuronal and muscle tissue, together with findings that thymopoietin is an endogenously occurring agent, could suggest that this immune-related polypeptide represents a ligand for the alpha-bungarotoxin receptors. The function of thymopoietin at the alpha-bungarotoxin receptor is as yet uncertain; however, a potential trophic, as well as other roles are suggested.     

Primary structure of an agonist binding subunit of the nicotinic acetylcholine receptor from bovine adrenal chromaffin cells

Activation by acetylcholine of a nicotinic acetylcholine receptor on the membrane of bovine chromaffin cells leads to membrane depolarization and to the subsequent triggering of catecholamine secretion. It is evident that acetylcholine receptors play a central role in the initial phase of the secretion process and, therefore, an extensive characterization of their molecular components and properties is of fundamental interest. With this intention, we have screened bovine adrenal medullary cDNA libraries with a probe coding for a fragment of the rat muscle acetylcholine receptor subunit. Several cDNA clones were isolated. The longest cDNA had an open reading frame encoding a 495-amino acid protein with a molecular weight of 56,911. The deduced primary structure contains features that indicate that the encoded protein is an or acetylcholine binding subunit, and, in fact, it manifests significant sequence similarity to previously cloned subunits. Sequence identity is particularly high with the 3 subunit, which is expressed in the rat pheochromocytoma PC12 cell line and in several brain areas, and consequently, it is considered a component of a neuronal acetylcholine receptor. Accordingly, the present results suggest that the agonist binding subunit of the nicotinic acetylcholine receptor from bovine chromaffin cells is an 3-type subunit, corroborating previous immunological and pharmacological evidence for the presence of a neuronal nicotinic receptor in chromaffin cells.Abbreviations used nAChR nicotinic acetylcholine receptor - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl and 0.015 M sodium citrate - kb kilobases - bp base pairs     

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.     

Nicotine receptors in the mammalian brain

Nicotine is a drug of abuse that presumably exerts its psychoactive effect through its interactions with nicotine binding sites in the central nervous system. Among its potential sites of action are the neuronal nicotinic acetylcholine receptors and the neuronal alpha-bungarotoxin binding sites. In this review we focus on the neuronal nicotinic acetylcholine receptors, their diversity, distribution, and functions as nicotine receptors or as mediators of synaptic transmission in the mammalian brain. We find that the complexity characteristic of the gene family encoding the subunits of these receptors is reflected both in the pattern of expression of the genes and in the pharmacological diversity of the expressed receptors.     

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.     

Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.     

A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors

alpha-Bungarotoxin, the classic nicotinic antagonist, has high specificity for muscle type alpha1 subunits in nicotinic acetylcholine receptors. In this study, we show that an 11-amino-acid pharmatope sequence, containing residues important for alpha-bungarotoxin binding to alpha1, confers functional alpha-bungarotoxin sensitivity when strategically placed into a neuronal non-alpha subunit, normally insensitive to this toxin. Remarkably, the mechanism of toxin inhibition is allosteric, not competitive as with neuromuscular nicotinic receptors. Our findings argue that alpha-bungarotoxin binding to the pharmatope, inserted at a subunit-subunit interface diametrically distinct from the agonist binding site, interferes with subunit interface movements critical for receptor activation. Our results, taken together with the structural similarities between nicotinic and GABAA receptors, suggest that this allosteric mechanism is conserved in the Cys-loop ion channel family. Furthermore, as a general strategy, the engineering of allosteric inhibitory sites through pharmatope tagging offers a powerful new tool for the study of membrane proteins.     

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.     

Antisera against an acetylcholine receptor alpha 3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh-binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of alpha 3 ACh-binding subunits expressed in bacteria, we show that ganglionic AChRs contain alpha 3 ACh-binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit-specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.