AlF4 - induced electrophysiological response and melatonin receptor expression in Xenopus oocytes

1 Injection of mRNA from the pars tuberalis (PT) of the ovine pituitary into Xenopus oocytes resulted in the expression of melatonin receptors within 2-4 days. 2 Melatonin had no effect on the membrane conductance of injected oocytes when applied by itself, but inhibited the Ca²⁺-dependent oscillatory Cl^- currents induced by phosphatidylinositol (PI) second messenger system activation. The PI system was stimulated in oocytes by application of AlF₄ ^-, a compound which binds to GDP at the nucleotide binding site on the G-protein, mimics GTP, and directly stimulates G-proteins, or via the endogenous muscarinic receptor. 3 The AlF₄ ^- response was activated at a range of membrane voltages, showed a reversal potential of -20 mV and was dose-dependent. Furthermore, the response was dependent on internal Ca²⁺, but modified by external Ca²⁺ and was only partially blocked by pertussis toxin. 4 Application of melatonin to PT mRNA-injected oocytes during carbachol or AlF₄ ^- induced response returned the membrane current to pre-stimulation baseline levels; the effect was dose-dependent and showed little voltage-dependence. Serotonin, N-acetyl-serotonin and dopamine showed little inhibitory action. However, iodomelatonin, an agonist with a high affinity for the melatonin receptor in the PT, inhibited the oscillations with a 10-fold greater potency than melatonin. 5 Fractionation of the mRNA on a 5-25% sucrose gradient limited the sequence encoding the melatonin receptor to fractions coincident with or just slightly ahead of the 28S rRNA peak.