Modulation of GABAA Receptor Function by G Protein-coupled 5-HT2C Receptors

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Abstract

Two classical neurotransmitters, 5-hydroxytryptamine (5-HT) and GABA, coexist in neurons of the medulla oblongata, and activation of 5-HT receptors modulates GABAA receptor function in neurons of the ventral tegmental area, substantia nigra and cerebellum. We now report that activation of 5-HT2C receptors produces a long-lasting (20–90 min) inhibition of GABAA receptors in Xenopus oocytes coexpressing both types of receptors. 5-HT2C receptors caused a ∼ 60% decrease in the GABAA receptor Emax without affecting the EC50 or Hill coefficient. Intracellular microinjection of 500 μM BAPTA blocked, whereas microinjection of inositol 1,4,5-triphosphate mimicked the inhibitory action of 5-HT2C receptors. The inhibition was independent of the GABAA receptors subunit composition; receptors containing α2β1, α1β1, α1β1γ2L, and α2β1γ2S were inhibited to the same extent by 5-HT2C receptor activation. Moreover, GABAA receptors composed of wild-type α2 plus mutant β1(S409A) subunits were inhibited to the same extent as wild-type receptors. The nonspecific protein kinase inhibitor, staurosporine, and the inhibitor of serine/threonine protein phosphatases, calyculin A, did not block the inhibitory effects of 5-HT2C receptors. The results with these inhibitors, taken together with those obtained with GABAA receptors with different subunit compositions, suggest that protein kinases or serine/threonine phosphatases are not involved in this GABAA receptor modulatory process. Thus, we propose that 5-HT2C receptors inhibit GABAA receptors by a Ca2+-dependent, but phosphorylation independent, mechanism and that 5-HT and GABA may act as cotransmitters to regulate neuronal activity. Furthermore, disruption of the cross-talk between these receptors may play a role in the anti-anxiety actions of 5-HT2 receptor antagonists. Copyright © 1996 Elsevier Science Ltd

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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