| Abstract No.: | C-B3036 |
| Country: | Canada |
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| Title: | ACTIVATION OF α7 NICOTINIC ACETYLCHOLINE RECEPTORS ATTENUATES CONSTITUTIVE INTERNALIZATION |
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| Authors/Affiliations: | 1 Kirk Young*; 2 R. Jane Rylett;
1 University of Western Ontario, London, ON, Canada; 2 University of Western Ontario, Robarts Research Institute, London, ON, Canada
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| Content: | Alpha 7 nicotinic acetylcholine receptors (α7nAChR) are excitatory ligand-gated ion channels, activated by the neurotransmitter acetylcholine, that are found in the peripheral and central nervous system. Α7nAChRs participate in fast neurotransmission and, through their high permeability to calcium, can modulate the release of other neurotransmitters and promote neuron survival. The neuroprotective signalling and regulatory effects of α7nAChRs upon neurotransmission are likely affected by the number and density of receptors expressed on the surface of neurons. This may be under the control of post-translational modifications that alter the endocytosis and trafficking of the α7nAChR protein.
Objectives: Investigate the kinetics of receptor endocytosis in the presence and absence of the receptor agonist, nicotine and the specific receptor antagonist, methyllycaconitine (MLA).
Materials and Methods: The human α7nAChR subunit was cloned from a cDNA library and a FLAG epitope tag was added to the extracellular C-terminus by PCR (FLAGα7nAChR). The FLAGα7nAChR cDNA was transfected into SH-SY5Y neuroblastoma cells to create a cell line that stably expressed FLAGα7nAChR protein (SH-FLAGα7R). To examine the internalization of receptors, surface proteins on SH-FLAGα7R cells were labelled with NHS-SS-biotin on ice, and the cells were subsequently incubated at 37°C for different time points in the presence or absence of 10 or 100 µM nicotine, with or without 10 µM MLA. The cells were returned to ice and the biotin labelling of non-internalized proteins was stripped with 2-mercaptoethanesulfonate. Biotinylated proteins were isolated from cell lysates by precipitation with NeutrAvidin resin and the presence of FLAGα7nAChR in biotinylated fractions was detected by immunoblotting with anti-FLAG antibody. Internalization of α7nAChR was quantified from immunoblots as a percentage of the total amount of biotinylated surface receptors from cells that were kept on ice and not stripped. To examine the effects of nicotine on the level of surface receptors, biotinylation was performed after incubating the cells at 37°C, without stripping.
Results: FLAGα7nAChRs constitutively internalized to a maximum of 50% within 5 min and this intracellular pool of biotinylated receptors declined with increasing incubation time. When treated with nicotine, the level of receptor internalization was reduced, to a minimum of 8%, but increased with longer incubation times. A concentration of 100 µM nicotine was more effective than 10 µm at reducing receptor internalization. Nicotine increased the level of receptors on the surface of cells with time. MLA attenuated the effects of nicotine both on receptor internalization and cell surface expression.
Conclusions: Nicotinic stimulation appears to retain α7nAChRs on the cell surface and reduces their rate of internalization, while acting to increase the total level of surface expression over time. Blocking receptor activation with MLA attenuates the effects of nicotine. |
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