Location of functional regions of acetylcholine receptor alpha-subunit by site-directed mutagenesis

The availability of cloned cDNAs encoding the four subunits of the Torpedo acetylcholine receptor, which can be expressed to make functional receptors in Xenopus oocytes, has made possible a detailed investigation of the functions of the different structural components of the receptor. The functional analysis of receptors with alpha-subunits altered at specific sites by site-directed mutagenesis of the cDNA has allowed the location of specific regions of the alpha-subunit molecule involved in acetylcholine binding and forming a transmembrane ionic channel.     

Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.     

Channel properties of an insect neuronal acetylcholine receptor protein reconstituted in planar lipid bilayers

A pentameric membrane protein composed of four types of polypeptide has been identified as the minimal structural unit responsible for the electrogenic action of acetylcholine on electrocytes and muscle cells. Because many populations of central and peripheral neurons also have nicotinic acetylcholine receptors (AChRs), considerable effort has recently gone into identifying the neuronal receptor. The central nervous tissue of insects contains very high concentrations of nicotinic AChRs, and we have recently purified an alpha-toxin binding protein, a putative AChR, from neuronal membranes of locusts. It is a component of high relative molecular mass, clearly composed of identical subunits, a structure predicted for an ancestral AChR protein. To verify that the purified polypeptides not only represent ligand binding sites but that they are indeed functional receptors, we have now reconstituted the isolated protein in a planar lipid bilayer. We show that in this system cholinergic agonists activate functional ion channels, that have properties comparable to those exhibited by the peripheral AChRs in vertebrates; thus, for the first time a functional acetylcholine receptor channel has been identified in nerve cells.     

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of alpha 2 beta gamma delta, and this is thought to be also the case for the mammalian skeletal muscle AChR. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four subunits of the Torpedo californica electroplax and calf muscle AChR and of the alpha- and gamma-subunits of the human muscle AChR; the primary structures of the gamma-subunit of the T. californica AChR and the alpha-subunit of the Torpedo marmorata AChR have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the epsilon-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the gamma-subunit than with the other subunits. cDNA expression studies indicate that the calf epsilon-subunit, as well as the calf gamma-subunit, can replace the Torpedo gamma-subunit to form the functional receptor in combination with the Torpedo alpha-, beta- and delta-subunits.     

Structural homology of Torpedo californica acetylcholine receptor subunits

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 1-3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4-6). We have recently elucidated the complete primary structures of the alpha-, beta- and delta-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides. Here, we report the whole primary structure of the gamma-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.     

Quaternary structure of the acetylcholine receptor

The five membrane-spanning subunits of the acetylcholine receptor have been resolved in electron microscope images and are shown to lie at pentagonally symmetrical positions around the channel over a large fraction of their length. The channel consists of a wide synaptic portion and a narrow portion extending through the membrane into the interior of the cell.     

Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor.

Ligand-gated ion channels, a major class of cell-surface proteins, have a pseudosymmetric structure with five highly homologous subunits arranged around a central ion pore. The correct assembly of each channel, whose subunit composition varies with cell type and stage of development, requires specific recognition between the subunits. Assembly of the pentameric form of the acetylcholine receptor from adult muscle (AChR; alpha 2 beta epsilon delta) proceeds by a stepwise pathway starting with the formation of the heterodimers, alpha epsilon and alpha delta. The heterodimers than associate with the beta subunit and with each other to form the complete receptor. We have now determined which parts of the subunits mediate the interactions during assembly of the adult form of the receptor from mouse muscle by using a chimaeric subunit in which the N-terminal and C-terminal extracellular domains are derived from the epsilon subunit with the remainder from the beta subunit. The epsilon and beta subunits were chosen because the epsilon subunit forms a heterodimer with the alpha subunit in the pathway for assembly of the receptor, whereas the beta subunit does not. The epsilon beta chimera can substitute for the epsilon but not the beta subunit in the oligomeric receptor, indicating that the alpha subunit specifically recognizes an extracellular domain of the epsilon subunit.     

Coexpression of two distinct muscle acetylcholine receptor alpha-subunits during development

The nicotinic acetylcholine receptor is a ligand-gated channel that mediates signalling at the vertebrate neuromuscular junction. It is a pentameric complex of four different subunits, assembled with a stoichiometry of alpha 2 beta gamma delta. Muscle-like alpha-subunits have been cloned from Torpedo, mouse, calf, rat, chicken, human and Xenopus, and only a single alpha-subunit complementary DNA from each species has been detected. We report here the cloning and characterization of a second muscle alpha-subunit cDNA from Xenopus, and show that this and a previously reported Xenopus alpha-subunit cDNA are encoded by distinct genes. The novel alpha-subunit reported here is expressed uniquely in oocytes; but both types of alpha-subunit are coexpressed throughout muscle development. This latter observation indicates that the expression of these two alpha-subunits is different from a previously reported developmental 'subunit-switch' mechanism used to generate channel diversity.     

Sequence and expression of a frog brain complementary DNA encoding a kainate-binding protein

Excitatory amino acids (EAAs) are important neurotransmitters in the vertebrate central nervous system. Electrophysiological and ligand-binding studies indicate that at least three different receptor subtypes for EAAs exist--N-methyl-D-aspartate, kainate and quisqualate receptor subtypes--on the basis of the preferred agonist of the receptors. We recently purified a kainate-binding protein (KBP) from frog (Rana pipiens berlandieri) brain by domoic acid (a high-affinity kainate analogue) affinity chromatography, and showed that the kainate-binding activity was associated with a protein of relative molecular mass 48,000 (Mr 48 K). The pharmacological properties and the anatomical distribution of KBP were consistent with those of a kainate receptor-ionophore complex. We have now isolated a complementary DNA encoding KBP of Mr 48 K. The deduced amino-acid sequence of the KBP has similar hydrophobic profiles to those found in other ligand-gated ion channel subunits, and shows some amino-acid sequence similarities to the corresponding regions of brain nicotinic acetylcholine receptor subunits. Localization of the KBP messenger RNAs by in situ hybridization histochemistry is compatible with the results of immunohistochemistry and receptor autoradiography studies. COS-7 cells transfected with the cDNA encoding the KBP show high-affinity kainate-binding activity with pharmacological properties similar to those of the biochemically purified KBP. These results provide the first molecular characterization of an EAA-binding site and raise the possibility that the KBP cDNA encodes a ligand-binding subunit of a kainate receptor-ionophore complex.     

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.     

Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit

We have isolated a complementary DNA clone containing sequences homologous to those encoding the alpha-subunit of a mouse muscle nicotinic acetylcholine receptor. Based on the structural similarities between the encoded protein and the muscle acetylcholine receptor alpha-subunit, and the presence of hybridizing RNA species in the brain, we propose that this clone codes for a neural nicotinic acetylcholine receptor alpha-subunit.     

Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.     

A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25

Lung cancer is the most common cause of cancer death worldwide, with over one million cases annually. To identify genetic factors that modify disease risk, we conducted a genome-wide association study by analysing 317,139 single-nucleotide polymorphisms in 1,989 lung cancer cases and 2,625 controls from six central European countries. We identified a locus in chromosome region 15q25 that was strongly associated with lung cancer (P = 9 x 10(-10)). This locus was replicated in five separate lung cancer studies comprising an additional 2,513 lung cancer cases and 4,752 controls (P = 5 x 10(-20) overall), and it was found to account for 14% (attributable risk) of lung cancer cases. Statistically similar risks were observed irrespective of smoking status or propensity to smoke tobacco. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits (CHRNA5, CHRNA3 and CHRNB4). Such subunits are expressed in neurons and other tissues, in particular alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines, and they bind to N'-nitrosonornicotine and potential lung carcinogens. A non-synonymous variant of CHRNA5 that induces an amino acid substitution (D398N) at a highly conserved site in the second intracellular loop of the protein is among the markers with the strongest disease associations. Our results provide compelling evidence of a locus at 15q25 predisposing to lung cancer, and reinforce interest in nicotinic acetylcholine receptors as potential disease candidates and chemopreventative targets.     

Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons

Glycine is an important inhibitory transmitter in the brainstem and spinal cord. In the trigeminal subnucleus caudalis (medullary dorsal horn) and in the spinal dorsal horn (the relaying centres for processing pain and sensory information), glycine inhibits the glutamate-evoked depolarization and depresses firing of neurons. The binding of glycine to its receptor produces a large increase in Cl- conductance, which causes membrane hyperpolarization. The selectivity and gating properties of glycine receptor channels have been well characterized; the glycine receptor molecules have also been purified. The amino-acid sequence, deduced from complementary DNA clones encoding one of the peptides (the 48K subunit), shows significant homology with gamma-aminobutyric acid A (GABAA) and nicotinic acetylcholine receptor subunits, suggesting that glycine receptors may belong to a superfamily of chemically gated channel proteins. However, very little is known about the modulation of glycine receptor channels. We have investigated the regulation of strychnine-sensitive glycine receptor channels by cyclic AMP-dependent protein kinase in neurons isolated from spinal trigeminal nucleus of rat and report here that the protein kinase A dramatically increased the glycine-induced Cl- currents by increasing the probability of the channel openings. GS protein, which is sensitive to cholera toxin, was involved in the modulation.     

Comparative and statistical analysis of nAChR sequences: An <Emphasis Type="Italic">ab initio</Emphasis> approach to the origin of molecular discrimination

Compared with traditional structure-based approaches for the identification of species-specific ligands, the ab initio approach, based on large-scale protein sequences from different species, has been used to locate specific sites that may be important to the molecular selectivity of species. Statistically significant differences in the distribution of residues in different species and differences in the physicochemical properties of residue-specific sites may largely account for species selectivity. The nicotinic acetylcholine receptor (nAChR), an important neuro-receptor with significantly different ligand selectivity in different species, was used to test our method. Because of the lack of nAChR structural information, the mechanism of ligand discrimination is unclear which hinders attempts at novel molecular design. In this study, the specific site residues 186 and 189 in the principal subunits and residues 34, 55, 56, 57, 106 and 112 in complementary subunits of nAChR were identified by applying our method with stringent statistical cutoffs. These sites were predicted to contribute to ligand selectivity and this result coincides well with the known experimental data.     

A new class of anthelmintics effective against drug-resistant nematodes

Anthelmintic resistance in human and animal pathogenic helminths has been spreading in prevalence and severity to a point where multidrug resistance against the three major classes of anthelmintics--the benzimidazoles, imidazothiazoles and macrocyclic lactones--has become a global phenomenon in gastrointestinal nematodes of farm animals. Hence, there is an urgent need for an anthelmintic with a new mode of action. Here we report the discovery of the amino-acetonitrile derivatives (AADs) as a new chemical class of synthetic anthelmintics and describe the development of drug candidates that are efficacious against various species of livestock-pathogenic nematodes. These drug candidates seem to have a novel mode of action involving a unique, nematode-specific clade of acetylcholine receptor subunits. The AADs are well tolerated and of low toxicity to mammals, and overcome existing resistances to the currently available anthelmintics.     

5-HT3 receptors mediate inhibition of acetylcholine release in cortical tissue

The release of cerebral acetylcholine from terminals in the cerebral cortex has been shown to be regulated by 5-hydroxytryptamine (5-HT) but it is not known which subtype of the 5-HT receptor is involved. 5-HT receptor agonists increase acetylcholine levels in vivo, indicating a reduced turnover, and reduce release of acetylcholine from striatal slices in vitro. Depleting 5-HT by inhibiting synthesis or by destroying the neurons containing 5-HT potentiates acetylcholine release, and increases acetylcholine turnover in the cerebral cortex and hippocampus. Selective antagonists for the 5-HT3 receptor subtypes which seem to have effects on mood and activity may exert their effect through the regulation of acetylcholine release in the cortex and limbic system. Radioligand binding studies show a high density of 5-HT3 receptors in the cholinergic-rich entorhinal cortex and we provide evidence that a reduction in cortical cholinergic function can be effected in vitro by 5-HT3 receptors.     

Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes

The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time.     

Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization

Recent studies have provided evidence for a role of protein phosphorylation in the regulation of the function of various potassium and calcium channels (for reviews, see refs 1, 2). As these ion channels have not yet been isolated and characterized, it has not been possible to determine whether phosphorylation of the ion channels themselves alters their properties or whether some indirect mechanism is involved. In contrast, the nicotinic acetylcholine receptor, a neurotransmitter-dependent ion channel, has been extensively characterized biochemically and has been shown to be directly phosphorylated. The phosphorylation of this receptor is catalysed by at least three different protein kinases (cyclic AMP-dependent protein kinase, protein kinase C and a tyrosine-specific protein kinase) on seven different phosphorylation sites. However, the functional significance of phosphorylation of the receptor has been unclear. We have now examined the functional effects of phosphorylation of the nicotinic acetylcholine receptor by cAMP-dependent protein kinase. We investigated the ion transport properties of the purified and reconstituted acetylcholine receptor before and after phosphorylation. We report here that phosphorylation of the nicotinic acetylcholine receptor on the gamma- and delta-subunits by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (ACh). These results provide the first direct evidence that phosphorylation of an ion channel protein modulates its function and suggest that phosphorylation of postsynaptic receptors in general may play an important role in synaptic plasticity.