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Ric-3 chaperone-mediated stable cell-surface expression of the neuronal alpha7 nicotinic acetylcholine receptor in mammalian cells.

ABSTRACT: AIM: Studies of the alpha7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of alpha7-type AChRs(1). The current work aims at obtaining and characterizing a cell line with high functional expression of the human alpha7 AChR. METHODS: Ric-3 cDNA was incorporated into SHE-P1-halpha7 cells expressing the alpha7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [(125)I]alpha-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent alpha-bungarotoxin, anti-alpha7 antibody, and GFP-alpha7 were performed on the new clone. RESULTS: The human alpha7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-halpha7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [(125)I]alphaBTX saturable binding with an apparent K(D) of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-halpha7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-alpha7 antibodies. In contrast, chaperone-less SHE-P1-halpha7 cells expressed only intracellular alpha7 AChRs and failed to produce detectable single-channel currents. CONCLUSION: The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal alpha7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

SUBMITTER: Valles AS

PROVIDER: S-EPMC4002380 | biostudies-other | 2009 Jun

REPOSITORIES: biostudies-other

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Project description:Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

Project description:BackgroundCigarette smoking adversely affects endochondral ossification during the course of skeletal growth. Among a plethora of cigarette chemicals, nicotine is one of the primary candidate compounds responsible for the cause of smoking-induced delayed skeletal growth. However, the possible mechanism of delayed skeletal growth caused by nicotine remains unclarified. In the last decade, localization of neuronal nicotinic acetylcholine receptor (nAChR), a specific receptor of nicotine, has been widely detected in non-excitable cells. Therefore, we hypothesized that nicotine affect growth plate chondrocytes directly and specifically through nAChR to delay skeletal growth.Methodology/principal findingsWe investigated the effect of nicotine on human growth plate chondrocytes, a major component of endochondral ossification. The chondrocytes were derived from extra human fingers. Nicotine inhibited matrix synthesis and hypertrophic differentiation in human growth plate chondrocytes in suspension culture in a concentration-dependent manner. Both human and murine growth plate chondrocytes expressed alpha7 nAChR, which constitutes functional homopentameric receptors. Methyllycaconitine (MLA), a specific antagonist of alpha7 nAChR, reversed the inhibition of matrix synthesis and functional calcium signal by nicotine in human growth plate chondrocytes in vitro. To study the effect of nicotine on growth plate in vivo, ovulation-controlled pregnant alpha7 nAChR +/- mice were given drinking water with or without nicotine during pregnancy, and skeletal growth of their fetuses was observed. Maternal nicotine exposure resulted in delayed skeletal growth of alpha7 nAChR +/+ fetuses but not in alpha7 nAChR -/- fetuses, implying that skeletal growth retardation by nicotine is specifically mediated via fetal alpha7 nAChR.Conclusions/significanceThese results suggest that nicotine, from cigarette smoking, acts directly on growth plate chondrocytes to decrease matrix synthesis, suppress hypertrophic differentiation via alpha7 nAChR, leading to delayed skeletal growth.

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Ric-3 chaperone-mediated stable cell-surface expression of the neuronal alpha7 nicotinic acetylcholine receptor in mammalian cells.

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