R. Adron Harris, Ph.D., Director, Waggoner Center for Alcohol and Addiction Research, Institute for Cellular and Molecular Biology, University of Texas at Austin, with colleagues Maria Paola Mascia, Ph.D. (University of Texas) and James R. Trudell, Ph.D. (Stanford University), developed a way to attach an anesthetic analogue called propanethiol to amino acid residues at a specific site in glycine and GABAA receptors. GABA and glycine receptors are the primary mediators of inhibitory neurotransmission in the brain and spinal cord.
The new work adds weight to previous studies that suggested that alcohols and anesthetic drugs exert some of their effects by interacting with specific protein molecules in the brain. Propanethiol (and also propyl-methanethiosulfanate), the researchers found, irreversibly enhances receptor function and obviates the ability of other alcohols and anesthetics to potentiate receptor function.
"Today's report advances general medical understanding of the basic pharmacology of alcohols and anesthetics," said Enoch Gordis, M.D., Director of the National Institute on Alcohol Abuse and Alcoholism (NIAAA). "Accumulating knowledge of how beverage alcohol (ethanol) produces its anesthetic and intoxicating effects at these receptors may lead to new pharmacologic and behavioral interventions."
For years, prevailing wisdom held that, unlike drugs with a single site of action, alcohols and anesthetics acted on many nonspecific sites of the neuronal membrane. More recent research has shown that these compounds act on specific receptor proteins, including the glycine and GABA neurotransmitter-activated ion channels. Defining the precise mechanisms of these actions, however, has defied traditional research methods, largely because of the low affinities and rapid kinetics that characterize alcohol and anesthetic compounds. To overcome these obstacles, Dr. Harris and his colleagues used anesthetic alcohol analogues capable of forming irreversible (covalent) bonds with specific amino acids.
Previous studies suggested that alcohols and general anesthetics interacted with the amino acids of the second (TM2) and third (TM3) transmembrane portions of the glycine and GABA receptors and that the presence of a serine amino acid (S267) in TM2 and an alanine amino acid (A288) in TM3 was necessary for the alcohols and anesthetics to trigger receptor response. To test whether either S267 and A288 in the glycine receptor and equivalent residues (S270 and A291) in the GABAA receptor indicated a critical binding site, Dr. Harris and his colleagues changed S267 and A288 to cysteine and tested whether propanethiol or proply-methanethiosulfanate bound with cysteine at the critical positions. If either S267 or A288 was a critical binding site, the research team reasoned, the analogue should irreversibly activate the altered receptors but reversibly activate the unchanged receptors. The researchers found this effect at a specific TM2 site in both glycine and GABAA receptor subunits.
"While other possible explanations cannot be fully ruled out, our results are extremely suggestive that the binding of alcohols and anesthetics in a protein cavity formed in part by a single amino acid is both necessary and sufficient to enhance receptor function," said Dr. Harris. "This indicates that anesthetics act by a mechanism closer to that of traditional receptor-mediated pharmacology than was previously thought. We believe this approach can help steer future research to define anesthetic binding sites on other brain proteins."
The NIAAA, the National Institute of General Medical Sciences, and the Texas Commission on Alcohol and Drug Abuse supported the research. For alcohol research information, please visit http://www.niaaa.nih.gov or telephone NIAAA Press (301/443-3860).
Reprints are available from the PNAS editorial office (tel. 202/334-2138). For additional information about the research, please telephone S.John Mihic, Ph.D. (tel. 512/232-7174) through July 31 or Adron Harris, Ph.D. (tel. 512/232-2514) after July 31.