ETHANOL AND PHOSPHORYLATION OF THE 5-HT3 RECEPTOR

The function of the 5-hydroxytryptamine3 (5-HT3) receptor is enhanced by alcohols, but the mechanism(s) of action remains unknown. In addition, regulation of the 5-HT3 receptor by post-translational modification events, such as phosphorylation, has not been well characterized. To date, one subunit of the 5-HT3 receptor has been cloned, and it possesses multiple consensus sequence sites for protein kinase dependent phosphorylaiton. Previous work with other ligand-gated ion channels, namely the GABA A and NMDA receptors, has suggested that protein kinase C may be involved in mediating these receptors' responsiveness to ethanol. Therefore, it is reasonable to speculate that kinases may modulate the responsiveness of the 5-HT 3 receptor to alcohols. In the present proposal, functional studies of the 5-HT3 receptor will be performed using Xenopus laevis oocytes as an expression system. Two-electrode voltage clamp electrophysiological recordings of expressed wild-type or mutagenized 5-HT3 receptors will be made. First, the sensitivity of the 5-HT3 receptor to alcohols will be measured. Next, the ability of serine/threonine kinases to alter 5-HT3 receptor function will be addressed. To determine which amino acid(s) are substrates for these kinases, candidate serines in consensus sequences for protein kinase dependent phosphorylation in the receptor will be mutated to nonphosphorylatable residues. Kinases which alter 5-HT3 receptor function will be examined for their ability to change the sensitivity of this receptor to alcohols. The alternative hypothesis, that tonic kinase activity underlies the 5-HT3 receptor's sensitivity to alcohols, will be tested by measuring alcohol modulation of 5-HT3 receptors with mutagenized consensus sequence sites for phosphorylation. Thus, the ultimate goal of the proposal is to determine if phosphorylation events alter the function of the 5-HT3 receptor and its modulation by alcohols. A better understanding of the regulation of the 5-HT3 receptor is important, given that this receptor may be involved in mediating the rewarding actions of drugs of abuse such as ethanol.