The molecular mechanisms of alcohol's actions in the nervous system are not known. This project combined molecular, biological and electrophysiological techniques to study the mechanisms of alcohol action on neural systems. The recombinant NMDA receptor subunits ~1, n1/~1, n2/~1, n3/~1, were expressed in Xenopus oocytes and their sensitivity to ethanol was investigations using the two-electrode voltage-clamp technique. NMDA-activated currents associated with n1/~1 or n2/~1 subunits, were significantly inhibited by 50 Mm ethanol, whereas NMDA currents associated with ~1 or n3/~1 subunits were not. Since NMDA receptor subunits are differentially distributed throughout the brain, the observations suggest that the differential sensitivity of NMDA receptor subunits to ethanol may contribute to the differences in ethanol sensitivity observed in different types of neurons. A possible role of phosphorylation in ethanol inhibition of NMDA-activated ion current was investigated using subunits n1/~1. Phorbol 12-myristrate 13-acetate (TPA), an activator of protein kinase C (PKC), increased the amplitude of NMDA current. After treatment with 1 uM TPA for 10 min, the inhibition of NMDA current by 100 Mm ethanol increased from 32% to 47%. Staurosporin, an inhibitor of PKC, had no significant effect on the inhibitory action of ethanol, but blocked the effect of TPA. The effect of ethanol was also studied on recombinant 5-HT3 receptors in Xenopus oocytes. Ethanol concentrations from 20 to 40 Mm potentiated, in a concentration-dependent manner, currents activated by 250 Nm or 500 Nm 5-HT; the EC50 was 126 Mm. With 1 Um or greater, potentiation of 5-HT current was not observed. The observations suggest that molecular biological techniques may be useful in elucidating the molecular mechanisms of alcohol's actions on neurotransmitter receptors.
