Immunological characterization and transmembrane topology of 5-hydroxytryptamine3 receptors by functional epitope tagging.
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.
Mukerji
J
J
Neurobiology Group, McGill University, Montreal, Quebec, Canada.
Haghighi
A
A
Séguéla
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D016895:Culture Media, Serum-Free; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D011985:Receptors, Serotonin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016324:Sequence Tagged Sites; D014018:Tissue Distribution; D014981:Xenopus
D016895:Culture Media, Serum-Free; D000939:Epitopes; D015335:Molecular Probes; D011985:Receptors, Serotonin