Structural determinants for high-affinity zolpidem binding to GABA-A receptors

The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the United States (Rush, 1998). Similar to classic benzodiazepines (BZDs), zolpidem binds at the extracellular N-terminal alpha/gamma subunit interface of the GABA-A receptor (GABAR). However, zolpidem differs significantly from classic BZDs in chemical structure and neuropharmacological properties. Thus, classic BZDs and zolpidem are likely to have different requirements for high-affinity binding to GABARs. To date, three residues -gamma 2Met57, gamma 2Phe77, and gamma 2Met130 -have been identified as necessary for high-affinity zolpidem binding (Proc Natl Acad Sci USA 94: 8824 -8829, 1997; Mol Pharmacol 52: 874 -881, 1997). In this study, we used radioligand binding techniques, gamma 2/alpha 1 chimeric subunits (chi), site-directed mutagenesis, and molecular modeling to identify additional gamma 2 subunit residues important for high-affinity zolpidem binding. Whereas alpha 1 beta 2 chi receptors containing only the first 161 amino-terminal residues of the gamma 2 subunit bind the classic BZD flunitrazepam with wild-type affinity, zolpidem affinity is decreased similar to 8-fold. By incrementally restoring gamma 2 subunit sequence, we identified a seven-amino acid stretch in the 2 subunit loop F region (amino acids 186-192) that is required to confer high-affinity zolpidem binding to GABARs. When mapped to a homology model, these seven amino acids make up part of loop F located at the alpha/gamma interface. Based on in silico zolpidem docking, three residues within loop F, gamma 2Glu189, gamma 2Thr193, and gamma 2Arg194, emerge as being important for stabilizing zolpidem in the BZD binding pocket and probably interact with other loop F residues to maintain the structural integrity of the BZD binding site.