Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in xenopus oocytes after cDNA injection

Ballivet, Marc; Nef, Patrick; Couturier, Sabine; Rungger, Duri; Bader, Charles; Bertrand, Daniel; Cooper, Ellis

doi:10.1016/0896-6273(88)90132-8

Cited by 125 publications

(53 citation statements)

References 30 publications

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“…At 24 h post-transfection, live cells were labeled with antibodies directed against the extracellular Nterminal domain of the ␣ 4 subunit to specifically label surfaceexpressed receptors. Since neither ␣ 4 nor ␤ 2 is capable of assembling into functional homo-oligomeric receptors (29), no surface labeling was observed when cells were mock-transfected or transfected with either ␣ 4 or ␤ 2 subunits alone (data not shown). However, when transfected together, mouse ␣ 4 and ␤ 2 subunits formed receptors that were detected at the surface of HEK293T cells by immunofluorescence with antibodies against the ␣ 4 (mAb 299) subunit (Fig.…”

Section: Resultsmentioning

confidence: 99%

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

Darsow

Booker

Piña‐Crespo

et al. 2005

Journal of Biological Chemistry

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The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed ␣ 4 ␤ 2 nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested. Neuronal nicotinic acetylcholine receptors (nAChRs)1 in mammalian brain compose a family of proteins encoded by 11 genes (␣ 2-7 , ␣ 9 -10 , and ␤ 2-4 ) that assemble into pentameric ligandgated ion channels (1-4). Although all combinations of subunits that form functional nicotinic receptors can bind nicotine, ␣ 4 -and ␤ 2 -containing receptors form the high affinity nicotine binding site (5-9) and are therefore thought to be the primary nAChR subtype affected by the relatively low nicotine concentrations (100 -500 nM) found in the blood of smokers (10).Chronic exposure of ␣4␤2 receptors to nicotine, as occurs in smokers, results initially in receptor desensitization, followed by subsequent up-regulation of high affinity nicotine binding sites (11). Upon nicotine withdrawal, the increased number of nAChRs recover from desensitization, resulting in excess activity in the nAChR system. It has been proposed that this cycle of nicotine-induced receptor up-regulation may contribute to the negative symptoms associated with nicotine withdrawal, resulting in continued tobacco consumption and, eventually, nicotine dependence (12).The mechanisms and cellular machinery required for nicotine-induced up-regulation remain unknown. However, a large body of research supports a consensus on several relevant points. First, up-regulation is observed in the brains of human smokers (13) as well as in chronically nicotine-treated animal models (8,14) and in cultured cells heterologously expressing nAChRs (15-18), suggesting that up-regulation requires basic, conserved cellular processes. It has been convincingly shown that increased nicotine binding reflects an increase in receptor number rather than receptor affinity (15, 19 -21), implying that up-regulation involves receptor protein dynamics rather th...

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Section: Resultsmentioning

confidence: 99%

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

Darsow

Booker

Piña‐Crespo

et al. 2005

Journal of Biological Chemistry

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“…Oocytes were seeded with their animal (brown) pole facing up so that the nucleus is located just underneath the cell membrane (24). This facilitated intranuclear injection of cDNA that has been described before (25). We used a semi-automated system, the Roboocyte (Multi Channel Systems, Reutlingen, Germany) whose features have been described elsewhere (26).…”

Section: Detection Of Phcl Splice Variants In Different Drosophila Stmentioning

confidence: 99%

A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons

Schnizler

Saeger

Pfeffer

et al. 2005

Journal of Biological Chemistry

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A systematic analysis of the Drosophila genome data reveals the existence of pHCl, a novel member of ligand-gated ion channel subunits. pHCl shows nearly identical similarity to glutamate-, glycine-, and histamine-gated ion channels, does however not belong to any of these ion channel types. We identified three different sites, where splicing generates multiple transcripts of the pHCl mRNA. The pHCl is expressed in Drosophila embryo, larvae, pupae, and the adult fly. In embryos, in situ hybridization detected pHCl in the neural cord and the hindgut. Functional expression of the three different splice variants of pHCl in oocytes of Xenopus laevis and Sf9 cells induces a chloride current with a linear current-voltage relationship that is inhibited by extracellular protons and activated by avermectins in a pH-dependent manner. Further, currents through pHCl channels were induced by a raise in temperature. Our data give genetic and electrophysiological evidence that pHCl is a member of a new branch of ligand-gated ion channels in invertebrates with, however, a hitherto unique combination of pharmacological and biophysical properties.Ligand-gated ion channels (LGICs) 1 mediate the fast inhibitory and excitatory responses of neuronal and muscle cells to neurotransmitters. A universal feature of the type of "Cys-loop" class of LGIC is a common topology of four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconservated cysteinebridge motive (1). In vertebrates this "Cys-bridge" family of phylogenetically related genes codes for cation channels activated by acetylcholine and serotonin or for anion channels activated by GABA and glycine (1). In addition, glutamateand serotonin-gated anion channel genes are known in invertebrates (2, 3). Recently, genes for histamine-gated chloride channels and GABA-gated cation channels were identified in invertebrates (4 -7). The molecular basis of further channel types like acetylcholine-gated chloride channels in invertebrates is, however, still unknown (8). Information from the Drosophila melanogaster genome sequencing project allows identifying all members of the superfamily of ligand-gated ion channels occurring in this species by bioinformatic analysis of new homologous genes. The summarized data obtained from several published bioinformatic analyses (5,6,9,10) show that the group of ligand-gated "chloride" channels consists of 12 genes that are coding for GABA, histamine, and glutamate receptors or new, homologous ion channel types. Four members of this group cannot be directly assigned to the GABA, glutamate, or histamine branches and thus code for putative new types of ligand-gated chloride channels with yet unknown function. In a systematic expression approach of these predicted novel types of ion channels in Xenopus oocytes, it was found that none of the typical neurotransmitters activated these novel types of channels (6). Therefore, we extended the molecular biological analysis of the mRNA and found that the gene CG6112 encodes fo...

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“…In addition, the availability of complementary DNA and mRNA allowed reconstitution of functional proteins in host systems such as Xenopus laevis oocytes or cell lines Barnard et al, 1982;Ballivet et al, 1988;Gopalakrishnan et al, 1995). Several genes encoding for nAChRs were identified from different invertebrate and vertebrate species.…”

Section: Introductionmentioning

confidence: 99%