René Anand

Assembly of Human Neuronal Nicotinic Receptor α5 Subunits with α3, β2, and β4 Subunits

Nicotinic acetylcholine receptors formed from combinations of α3, β2, β4, and α5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human α3, β2, β4, and α5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of α3β2, α3β4, α3β2α5, and α3β4α5 subunits but not for other combinations of one, two, or three of these subunits. α5 subunits assembled inside the oocyte with β2 but not with α3 subunits or other α5 subunits. α5 subunits coassembled very efficiently with α3β2 or α3β4 combinations. The presence of α5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also α3 and β2 or α3 and β4 subunits. The presence of α5 subunits increased the rate of desensitization of both receptors containing also α3 and β2 or α3 and β4 subunits. In the case of receptors containing α3 and β4 subunits, the addition of α5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing α3 and β2 subunits, the addition of α5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 μM and the EC50 for nicotine from 6.8 to 1.9 μM, while increasing the efficacy of nicotine from 50% on α3β2 receptors to 100% on α3β2α5 receptors. Both α3β2 and α3β2α5 receptors expressed in oocytes sedimented at the same 11 S value as native α3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma α3, β2, and α5 subunits were co-assembled, and 56% of the receptor subtypes containing α3 subunits also contained β2 subunits. The β2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from α3 and β2 or α3, β2, and α5 subunits rather than the low affinity exhibited by receptors formed from α3 and β4 or α3, β4, and α5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing α3 subunits in SH-SY5Y cells. The affinities of α3β2 receptors expressed in oocytes were similar to the affinities of native α3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.

Chapter 10 Structure and function of neuronal nicotinic acetylcholine receptors

Publisher Summary This chapter discusses some aspects of neuronal acetylcholine receptors (AChRs), reviews some of the recent studies of the mechanism by which chronic exposure to nicotine affects aα4β2 AChRs, and describes some of the pharmacological properties of neuronal AChRs, especially the differences between α7 and α8 AChRs and the usefulness of epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.l]-heptane) as a ligand for many types of neuronal AChRs. The basic homologies in structure of receptors in this superfamily have been illustrated in the chapter. Homologies in overall domain relationships are demonstrated by showing that functional mosaics could be made in which the large N-terminal extracellular domain of α7 AChRs could be grafted just before the first transmembrane domain to the C-terminal part of 5HT 3 receptors. This resulted in receptors with acetylcholine-gated cation channels having the ion selectivity of 5HT 3 receptors. Close similarities in the overall structures of the ion channels are demonstrated by showing that changing only three amino acids in the sequence lining the cation-specific channel of excitatory α7 neuronal AChRs to amino acids typical of the anion-specific channels of inhibitory glycine or GABA A receptors changed the ion selectivity of the mutated a7 AChR channels from cations to anions

Homomeric and native α7 acetylcholine receptors exhibit remarkably similar but non-identical pharmacological properties, suggesting that the native receptor is a heteromeric protein complex

Sucrose gradient analysis of chick acetylcholine receptor (AChR) α7 subunits expressed in oocytes indicates that they form pharmacologically active homomers of the same size as native α7 AChRs, a size compatible with a complex of five α7 subunits. By immunoisolating the [35S]methionine‐labeled α7 subunits we also demonstrate that they do not appear to assemble with endogenous Xenopus AChR subunits. Pharmacological characterization of detergent‐solubilized brain α7 AChRs and α7 homomers reveals that they have similar but nonidentical properties. The pharmacological difference is most accentuated for cytisine (~50‐fold). Thus, at least in E18 chicken brain, most or all of the native α7 AChRs do not appear to be homomeric.

Determination of amino acids critical to the main immunogenic region of intact acetylcholine receptors by in vitro mutagenesis

The main immunogenic region (MIR) of the acetylcholine receptor (AChR) is the target for the majority of high‐affinity autoantibodies produced in myasthenia gravis patients. Some monoclonal antibodies (mAbs) to the MIR bind specifically, but with low affinity, to synthetic AChR α subunit peptides with the sequence α67–76. Studies of synthetic peptides suggest that amino acids α68 and α71 may be especially important to the antigenic structure of the MIR. We have studied the contribution of amino acids α68 and α71 to the antigenicity of the MIR on intact AChR by replacing α68 (N) and α71 (D) of Torpedo AChR α with α68 (D) and α71 (K) by site‐directed mutagenesis, expressing the mutated transcripts in Xenopus oocytes along with wild‐type Torpedo β,γ andδ subunits, and analyzing the expressed AChR for the binding of mAbs to the MIR. These mutations of the MIR greatly diminished binding of mAbs to the MIR. Thus, both α68 and α71 are critical to the antigenicity of the MIR in intact AChRs.

Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains.

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression inXenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for α-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the α7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length α7 AChR.

N-Methyl-d-aspartate receptor mediated toxicity in nonneuronal cell lines: characterization using fluorescent measures of cell viability and reactive oxygen species production

Cells transfected with specific N-methyl-D-aspartate (NMDA) receptor subtypes undergo cell death that mimics glutamate-induced excitotoxicity pharmacologically. We have further characterized the mechanisms of cell death resulting from NMDA receptor activation in such cells through development of cell counting methods based on co-transfection with green fluorescent protein. When co-transfected with NMDA receptors, GFP expression was limited to live cells as indicated by the observation that GFP was only detected in cells which were positive for markers of live cells, and was found in no cells which were trypan blue or propidium iodide positive. Using co-transfection with green fluorescent protein and cell counting of viable cells with a fluorescence activated cells sorter, we confirmed the subunit-specific profile of NMDA receptor-mediated cell death in cells transfected with NMDA receptors. Toxicity was greatest in the NR1A/2A receptor, less in the NR1A/2B receptor, and least in NR1A/2C receptors. Cell death also differed pharmacologically between subunit combinations. Cell death in cells transfected with NR 1A/2A was blocked by amino-phosphonovaleric acid at lower concentrations than in cells transfected with NR 1A/2B. In cells transfected with the NR1A/2A or NR1A/2B combinations but not NR1A/2C, cell death was also associated with production of reactive oxygen species. In addition, removal of the final 400 amino acids of the C-terminal region of NR2A decreased cell death. The use of GFP based cell counting provides a sensitive mechanism for assessing the mechanism of excitotoxicity in transfected cell models.

Chromosomal localization of seven neuronal nicotinic acetylcholine receptor subunit genes in humans

We have determined the chromosomal location of seven human neuronal nicotinic acetylcholine receptor subunit genes by genomic Southern analysis of hamster/human somatic cell hybrid DNAs. The beta 2 subunit gene was localized to human chromosome 1, the alpha 2 and beta 3 subunit genes were localized to human chromosome 8, the alpha 3, alpha 5, and beta 4 subunit genes were localized to human chromosome 15, and the alpha 4 subunit gene was localized to human chromosome 20. Mapping of the beta 2 subunit gene to chromosome 1 establishes a syntenic group with the amylase gene locus on human chromosome 1 and mouse chromosome 3, while mapping of the alpha 3 subunit gene to chromosome 15 confirms the existence of a syntenic group with the mannose phosphate isomerase gene locus on human chromosome 15 and mouse chromosome 9.

The nicotinic acetylcholine receptor gene family: structure of nicotinic receptors from muscle and neurons and neuronal alpha-bungarotoxin-binding proteins.

Three branches of the ligand-gated ion channel gene superfamily encode proteins that bind cholinergic ligands: 1) nicotinic acetylcholine receptors (AChRs) from skeletal muscle, 2) nicotinic AChRs from neurons, and 3) α-bungarotoxin-binding proteins (αBgtBPs) from neurons. AChRs from vertebrate muscles and nerves differ in subunit composition, and in some cases in functional role, but both appear to be formed from several homologous subunits which form ACh-gated cation channels. αBgtBPs from vertebrate neurons have uncertain subunit compositions, uncertain endogenous ligands, and unknown functions. The ligand-gated ion channel gene superfamily also includes receptors for GABA and glycine, which are ligand-gated anion channels, and it probably also includes other ligand-gated ion channels (Barnard et al., 1987; Betz and Becker, 1988). The relation of glutamate receptors to this superfamily is less certain (Gregor et al., 1989; Hollmann et al., 1989; Wada et al., 1989).

Neuronal Nicotinic Receptor Structure and Function

The best characterized subtype from the branch of the neuronal nicotinic receptor (nAChR) gene family which does not bind α-bungarotoxin has the subunit stoichiometry (α4)2(β2)3 and accounts for >90% of the high affinity nicotine binding in mammalian brains. Chronic treatment of cells transfected with α4β2 AChRs causes an increase in the amount of AChR with pharmacological characteristics and a time course that parallel the nicotine-induced increase in brain α4β2 AChRs. This is the result of a decrease in turnover of surface AChRs. Many of these surface AChRs are permanently functionally inactive. The predominant subtype of the branch of the neuronal AChR gene family which binds α-bungarotoxin (α-BTX) contains α7 subunits. α7 AChRs predominate in brain, while α8 AChRs predominate in retina, and α7α8 AChRs are a minor component in both tissues. α7 and α8 homomers have similar channel properties, but α8 homomers and native α8 AChRs have lower affinity for α-BTX and higher affinity for small cholinergic ligands than do α7 homomers and α7 AChRs. The high Ca++ permeability and rapid desensitization of α7 and α8 AChRs may enable them to participate in unusual synaptic mechanisms.