The primary goal of the proposed research is to probe structure- function relationships within the serotonin type 1 (5HT1) receptor agonist binding site. To achieve this goal, efforts will be made to unambiguously identify the neurotransmitter binding site by constitutively activating the receptor at the serotonin binding site. The ultimate goal of this research is to use these constitutively active receptors to study the long-term effects of 5HT1 receptor activation. This proposal is specifically focused on the incorporation of unnatural amino acids into 5HT1A receptors and their expression in intact cells. The following are the research objectives: l. Design and synthesis of unnatural amino acids that include serotonin itself in their side chains. 2. Incorporation of these unnatural amino acids into the putative 5HT1A agonist binding site using the nonsense codon suppression method for unnatural amino acid incorporation in Xenopus oocytes. 3. Identification of functional 5HT1A receptors through electrophysiological analysis of functionally coupled, coexpressed guanine binding protein (G-protein) activated, inwardly rectifying potassium (K+) channels.
