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. However, the molecular basis for alcohol modulation of 5-HT3 receptor function has not been determined. Previously, using a chimeric nicotinic-serotonergic receptor, we found evidence that alcohol effects on receptor function involve the extracellular N-terminal domain of the receptor (Mol Pharmacol 1996;50:1010-1016). We have been studying the effect of single amino acid mutations in the N-terminal domain of the 5-HT3 receptor on the function and ethanol sensitivity of the receptor and found that arginine 222 (R222) in the N-terminal domain of the 5-HT3 receptor is a molecular determinant of alcohol sensitivity of the receptor (J Biol Chem 2002;277:46256-46264). Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels, such as 5-HT3 receptors. However, the transduction mechanisms that link agonist binding to channel gating for these receptors remains poorly understood. Arginine 222 (R222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3 receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of R222 in the function of 5-HT3 receptors, we mutated the arginine residue to alanine (A) and expressed both the wild-type and the mutant receptor in HEK 293 cells. Functional studies of expressed wild-type and mutant receptors, using whole-cell patch-clamp recording, revealed that the R222A mutation increased the potency of the full agonist, 5-HT, and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization, but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue R222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3 receptors. Experiments are also in progress to elucidate the cellular mechanisms of alcohol and neuroactive substance action on neurotransmitter receptors such as NMDA receptors, GABA-A receptors, nicotinic acetylcholine (nACh) receptors and ATP receptors. These studies hold the promise that such physiological approaches will advance our knowledge of the cellular mechanisms of alcohol and neuroactive substance action in the nervous system and provide a foundation for understanding the cellular basis of alcohol abuse and alcoholism.
