Ethanol potentiates the function of serotonin type 3 (5-HT3) receptors in various types of neurons and several studies have suggested that 5-HT3 receptors may be involved in alcohol preference and reward mechanisms. We are investigating the molecular mechanism of ethanol action on 5-HT3 receptors and the molecular mechanisms involved in the modulation of 5-HT3 receptor function. In view of the ethanol sensitivity of 5-HT3 receptors and the possible involvement of 5-HT3 receptors in alcohol preference and reward mechanisms, it is important to elucidate the mechanisms involved in the modulation of 5-HT3 receptor function. In a previous study, we found that protein kinase C (PKC) activation potentiates 5-HT3 receptor-mediated responses (Neuroreport 6: 1464-1468, 1995). More recently we have been studying the molecular mechanisms involved in that modulation of 5-HT3 receptor function. We have found that the potentiation of 5-HT3 receptor-mediated responses by PKC activation does not appear to be due to direct phosphorylation of the 5-HT3 receptor protein because the potentiation is not affected by point-mutation of the putative sites for PKC phosphorylation. In addition, using a specific fluorescent-labeled antibody directed to the extracellular N-terminal domain of the 5-HT3 receptor, treatment with the PKC activator, 4-beta-phorbol 12-myristate 13-acetate (PMA), significantly increases surface immunofluorescence. PMA also increases the amount of 5-HT3 receptor protein in the cell membrane without affecting the amount receptor protein in the total cell extract. The magnitude of PMA potentiation of 5-HT3 receptor-mediated responses is correlated with the magnitude of PMA enhancement of the receptor abundance in the cell surface membrane. In addition, preapplication of phalloidin, which stabilizes the actin polymerization, significantly inhibits PMA potentiation of 5-HT3 receptor-mediated responses. On the other hand, latrunculin-A, which destabilizes actin cytoskeleton, enhances PMA potentiation of 5-HT3 receptor-mediated responses. The observations suggest that PKC modulates 5-HT3 receptor function and trafficking through an F-actin dependent mechanism. Experiments are also in progress to elucidate the molecular determinants for alcohol and neuroactive substance sensitivity of other neurotransmitter-gated membrane ion channels, such as N-methyl-D-aspartate (NMDA) receptors, gamma-aminobutyric type A (GABA-A) receptors, nicotinic acetylcholine (nACh) receptors, glycine receptors and ATP receptors. These studies hold the promise that such molecular biological approaches will advance our knowledge of the molecular basis of alcohol and neuroactive substance action in the nervous system and provide a foundation for understanding the molecular basis of alcohol abuse and alcoholism.
