Immunological Characterization and Transmembrane Topology of 5-Hydroxytryptamine3 Receptors by Functional Epitope Tagging |
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Authors: | Jishnu Mukerji,Ali Haghighi, Philippe Sé gué la |
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Affiliation: | Neurobiology Group, Montreal Neurological Institute, and; Department of Physiology, McGill University, Montreal, Quebec, Canada |
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Abstract: | Abstract: 5-Hydroxytryptamine3 (5-HT3) receptors are the only known monoamine receptors mediating fast excitatory responses in mammalian neurons. Their primary structure as well as their electrophysiological and pharmacological properties show a phylogenetic relation to nicotinic acetylcholine, GABAA, and glycine receptors. As a prototypical member of this gene superfamily, we investigated the membrane topology of functional homomeric 5-HT3 receptors by using epitope tagging of the channel subunits expressed in heterologous systems. Visualization of 5-HT3 receptors in transfected COS-7 cells, either in western blot (molecular mass 61.2 ± 0.8 kDa) or in situ, was performed with previously characterized antibodies recognizing artificial epitopes as well as with anti-fusion protein antibodies directed against a wild-type receptor intracellular domain. The extracellular location of the distal C-terminal tagged domain demonstrates the presence of a fourth transmembrane domain in 5-HT3 serotonin-gated channels. In this region, the significant homology between members of this class of neurotransmitter-gated channels suggests strongly that they have a common transmembrane organization basically different from glutamate-gated and ATP-gated channels. |
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Keywords: | 5-Hydroxytryptamine3 receptors Monoamine Xenopus oocyte Flag Immunofluorescence |
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