The function of strychnine-sensitive glycine receptors is enhanced in the presence of clinically relevant concentrations of ethanol and longer chain alcohols as well as volatile anesthetics. Since glycine receptors are mediators of inhibition in the spinal cord and in some areas of the brain, they may be involved in the sedative, anti-araiety and anesthetic effects of alcohol. Site-directed mutagenesis techniques have identified residues in transmembrane (TM) domains two (S267) and three (A288) that mediate the effects of alcohols and anesthetics. By mutating target S267 to cysteine, an alkane thiol anesthetic was able to covalently label the binding site and irreversibly enhance the function of the receptor. The following proposal stems from this work and includes three main aims. First, the question of whether transmembrane (TM) 2 residue S267 and TM3 residue A288 face one another in three- dimensional space to form an alcohol and volatile anesthetic binding pocket will be addressed. The methods will include biochemical analysis and site-directed mutagenesis in Xenopus laevis oocytes, Second, the volume of this binding pocket will be examined during channel gating for A288 and a region of amino acids in TM2. Lastly, amino acid residues in TM I and TM4, predicted to be in the vicinity of the putative alcohol/anesthetic binding pocket, will be tested to see if they participate in alcohol/anesthetic binding. The answers to these questions will provide information to more accurately model the alcohol/ anesthetic binding site in the glycine receptor (and other ligand-gated ion channels) and contribute to a better understanding of the mechanism by which drugs bind to and affect this receptor.
|Lobo, Ingrid A; Trudell, James R; Harris, R Adron (2006) Accessibility to residues in transmembrane segment four of the glycine receptor. Neuropharmacology 50:174-81|
|Lobo, Ingrid A; Harris, R Adron (2005) Sites of alcohol and volatile anesthetic action on glycine receptors. Int Rev Neurobiol 65:53-87|
|Lobo, Ingrid A; Mascia, Maria Paola; Trudell, James R et al. (2004) Channel gating of the glycine receptor changes accessibility to residues implicated in receptor potentiation by alcohols and anesthetics. J Biol Chem 279:33919-27|
|Lobo, Ingrid A; Trudell, James R; Harris, R Adron (2004) Cross-linking of glycine receptor transmembrane segments two and three alters coupling of ligand binding with channel opening. J Neurochem 90:962-9|