Structural Requirements for Eszopiclone and Zolpidem Binding to the γ-Aminobutyric Acid Type-A (GABA_A) Receptor Are Different

The sleep-aids zolpidem and eszopiclone exert their effects by binding to and modulating γ-aminobutyric acid type-A receptors (GABAARs), but little is known about the structural requirements for their actions. We made 24 cysteine mutations in the benzodiazepine (BZD) binding site of α1β2γ2 GABAARs and measured zolpidem, eszopiclone, and BZD-site antagonist binding. Mutations in γ2loop D and α1loops A and B altered the affinity of all ligands tested, indicating that these loops are important for BZD pocket structural integrity. In contrast, γ2loop E and α1loop C mutations differentially affected ligand affinity, suggesting that these loops are important for ligand selectivity. In agreement with our mutagenesis data, eszopiclone docking yielded a single model stabilized by several hydrogen bonds. Zolpidem docking yielded three equally populated orientations with few polar interactions, suggesting that unlike eszopiclone, zolpidem relies more on shape recognition of the binding pocket than on specific residue interactions and may explain why zolpidem is highly α1- and γ2-subunit selective.