Modulation of GABAA Receptor Function by G Protein-coupled 5-HT2C Receptors
Section snippets
In vitro transcription of cRNAs and microinjection into Xenopus oocytes
The human GABAA receptor subunits cDNAs were cloned on the eukaryotic expression vector pCDM8 (Invitrogen Corp., San Diego, CA, U.S.A.); the cloning of these subunits is described elsewhere (Hadingham et al. (1993a), Hadingham et al. (1993b); Wafford et al., 1993). Rat 5-HT2C-R cDNA was generously provided by Dr David Julius (Department of Pharmacology, UC San Francisco, U.S.A.); cRNAs were synthesized in vitro by using the mRNA capping kit from Stratagene cloning systems (La Jolla, CA,
Effects of 5-HT2C-R activation on GABA-activated Cl− currents
Bath application of 5-HT to Xenopus oocytes micro-injected with 5-HT2C cRNA produced inward currents (Fig. 1, upper panel) with an EC50 100 nM. These currents were abolished by microinjection of 500 μM BAPTA and consequently, correspond to Ca2+-activated Cl− currents (Takahashi et al., 1987; Sanna et al., 1994). In oocytes coexpressing 5-HT2C-Rs and GABAA-R (composed of α2β1 subunits), application of 0.1 μM 5-HT did not produce any appreciable effects on GABA-gated currents (Fig. 1, lower
DISCUSSION
The major finding of this study is that a brief activation of 5-HT2C-Rs produces a long-lasting inhibition of GABAA-R function in Xenopus oocytes coexpressing both types of receptors. The inhibitory effect of 5-HT2C-R depended on the concentration of 5-HT used to activate the receptors and was characterized by a decrease in the GABA Emax with no change in the GABA EC50 or Hill coefficient. The 5-HT2C-R-induced inhibition of GABAA-Rs was: (1) independent of subunit composition; (2) blocked by
Acknowledgements
This work has been supported by a fellowship (for a sabbatical leave) from Fundación Andes, Chile (J.P.H-T), a NRSA Grant AA05399 (C.F.V), and by NIAAA Grant AA06399 and the Department of Veterans Affairs (R.A.H.). We are grateful to Dr David Julius (Department of Pharmacology, UC San Francisco, U.S.A.) for kindly providing 5-HT2C cDNA, to Dr Tina Machu for synthesis of cRNA, to Paul Whiting (MSD Labs, Essex, U.K.) for GABA cDNAs and to Virgina Bleck for assistance with the figures. We thank
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