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.     

The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: beta 4

A new nicotinic acetylcholine receptor (nAChR) subunit, beta 4, was identified by screening a rat genomic library. In situ hybridization histochemistry revealed expression of the beta 4 gene in the medial habenula of adult rat brains. The primary structure of this subunit was deduced from a cDNA clone isolated from a PC12 cDNA library. Functional nAChRs were detected in Xenopus oocytes injected in pairwise combinations with in vitro synthesized RNAs encoding beta 4 and either the alpha 2, alpha 3, or alpha 4 subunit. Unlike the alpha 3 beta 2 receptor, the alpha 3 beta 4 receptor is not blocked by bungarotoxin 3.1, indicating that the beta subunit can affect the sensitivity of neuronal nAChRs to this toxin. These results extend the functional diversity of nicotinic receptors in the nervous system.     

Distribution of alpha7 and alpha4 nicotinic acetylcholine receptor subunits in several tissues of Triturus carnifex (Amphibia, Urodela)

The distribution of neuronal and non-neuronal mRNAs for alpha7 and alpha4 nicotinic acetylcholine receptor subunits was investigated in Triturus carnifex tissues using the in situ hybridization approach. The findings reveal a composite pattern of expression only partially overlapping for the two subunits; subunit alpha7 seems to be expressed widely throughout nervous, gastrointestinal and skin tissues, while alpha4 is present in a restricted number of cells of nervous and gastrointestinal tissue. We also found a specific pattern for each subunit; alpha7 and alpha4 associated exclusively to the epidermal glands and hypophysis, respectively; this is probably due to alternative roles that nicotinic acetylcholine receptors play in regulating physiological functions of non-neuronal amphibian tissues, rather than as mere neurotransmitters in the nervous system.     

Amino acid determinants of alpha 7 nicotinic acetylcholine receptor surface expression

Transient transfection has not been a successful method to express the alpha7 nicotinic acetylcholine receptor such that these receptors are detected on the cell surface. This is not the case for all ligand-gated ion channels. Transient transfection with the 5-hydroxytryptamine type 3 subunit cDNA results in detectable surface receptor expression. Cell lines stably expressing the alpha7 nicotinic acetylcholine receptor produce detectable, albeit variable, levels of surface receptor expression. alpha7 nicotinic acetylcholine receptor surface expression is dependent, at least in part, on cell-specific factors. In addition to factors provided by the cells used for receptor expression, we hypothesize that the surface expression level in transfected cells is an intrinsic property of the receptor protein under study. Employing a set of alpha7-5-hydroxytryptamine type 3 chimeric receptor subunit cDNAs, we expressed these constructs in a transient transfection system and quantified surface receptor expression. We have identified amino acids that control receptor distribution between surface and intracellular pools; surface receptor expression can be manipulated without affecting the total number of receptors. These determinants function independently of the cell line used for expression and the transfection method employed. How these surface expression determinants in the alpha7 nicotinic acetylcholine receptor might influence synaptic efficacy is discussed.     

Structure-function relationships in nicotinic acetylcholine receptors

1. A combination of molecular, biochemical, electrophysiological and immunological approaches has begun to resolve some of the questions about structure-function relationships of nicotinic acetylcholine receptors (AchRs). 2. Current structural studies suggest that models of the subunits which propose four transmembrane domains are correct. 3. It is also probable that the carboxy termini of the subunits are extracellular, while the putative amphpathic helix is intracellular. 4. Electrophysiological and ligand-binding experiments suggest that the M2 region forms the wall of the ion channel. 5. We have isolated clones from PC12 and rat brain cDNA libraries which we have shown, by functional expression, code for members of a gene family of nicotinic acetylcholine receptor subunits. 6. In situ hybridization studies have shown that the neuronal receptor subunit mRNAs are expressed in the mammalian central nervous system. 7. The muscle and neuronal nicotinic AchR subtypes we have expressed show differences in their pharmacological properties. 8. The isolation and identification of clones which code for receptors and voltage-activated ion channels will help in the understanding of a variety of disease states and assist in the design of drugs which are specific for unique molecular targets.     

Inflammatory cytokines decrease the expression of nicotinic acetylcholine receptor during the cell maturation

It is known that the nervous system significantly attenuates systemic inflammatory responses through the parasympathetic nervous system. Furthermore, it has been reported that the alpha7 subunit of a nicotinic acetylcholine receptor is required for a cholinergic inhibition against cytokine synthesis in a macrophage. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses and the maintenance of immunity, in this study, we investigated the expression level of nicotinic receptors of a p53-deficient APC cell line (JawsII) derived from a mouse bone marrow. We showed that stimulation of the JawsII cells with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) led increase of CD80 and CD86 expression while diminishment of the surface nicotinic receptor. On the other hand, stimulation of nicotinic receptor had no effect on these phenomena. Furthermore, we examined the ability of the cells to release cytokine when stimulated with both nicotine and LPS and showed that the stimulation with LPS augmented the secretion of IL-1a, IL-1b, IL-6, and TNF-α. These results suggested that nicotinic stimulation had no effect on the diminishment of alpha7 nicotinic acetylcholine receptor on JawsII cells by LPS stimulation.     

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.     

Beta 3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain

Screening of a rat brain cDNA library with a radiolabeled probe made from an alpha 3 cDNA (Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemanns, S., and Patrick, J. (1986) Nature 319, 368-374) resulted in the isolation of a clone whose sequence encodes a protein, beta 3, which is homologous (40-55% amino acid sequence identity) to previously described neuronal nicotinic acetylcholine receptor subunits. The encoded protein has structural features found in other nicotinic acetylcholine receptor (nAChR) subunits. Two cysteine residues that correspond to cysteins 128 and 142 of the Torpedo nAChR alpha subunit are present in beta 3. Absent from beta 3 are 2 adjacent cysteine residues that correspond to cysteines 192 and 193 of the Torpedo subunit. In situ hybridization histochemistry, performed using probes derived from beta 3 cDNAs, demonstrated that the beta 3 gene is expressed in the brain. Thus, beta 3 is the fifth member of the nAChR gene family that is expressed in the brain. The pattern of beta 3 gene expression partially overlaps with that of the neuronal nAChR subunit genes alpha 3, alpha 4, or beta 2. These results lead us to propose that the beta 3 gene encodes a neuronal nAChR subunit.     

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.     

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.     

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.     

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     

α7nAChR在上皮组织的表达及其功能的研究进展

α7 nAChR是配体门控离子通道蛋白超家族的典型代表,烟碱型乙酰胆碱受体的一个重要亚型,是复杂的五聚体跨膜蛋白,介导Na^＋、Ca^2＋流入,K^＋流出,尤以对Ca^2＋通透性高。α7 nAChR分布广泛且功能多样,不仅分布于中枢和外周神经系统,介导神经元的快速突触传递,其在许多非神经元细胞和组织中亦有表达,包括内皮细胞,支气管上皮细胞,皮肤角蛋白细胞,膀胱上皮细胞,血管平滑肌等,并参与其功能调节及功能障碍相关疾病的病理生理过程,如可调节细胞质运动和细胞间黏附,细胞增殖,血管生成以及肿瘤的侵袭和迁移。本文主要介绍烟碱型乙酰胆碱受体α7 亚型在不同胚层来源的上皮组织细胞中的表达及其功能特征,以期通过激活或抑制α7 nAChR的表达来降低与其密切相关疾病的发生率。     

Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.