cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.
AUTOR(ES)
Lübbert, H
RESUMO
We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=305079Documentos Relacionados
- Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes.
- Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes.
- 5-HT1c receptor is a prominent serotonin receptor subtype in the central nervous system.
- Chinese hamster serotonin (5-HT) type 2 receptor cDNA sequence.
- Serotonin inhibits Ca2+ currents in porcine melanotrophs by activating 5-HT1C and 5-HT1A receptors.
