The role of loops B and C in determining the potentiation of GABA(A) receptors by midazolam

Many benzodiazepines are positive allosteric modulators (PAMs) of GABA(A) receptors that cause sedation, hypnosis, and anxiolysis. Benzodiazepines bind GABA(A) receptors at the extracellular interface of the α and γ subunits. Within the α subunit, the benzodiazepine binding site is defined by three highly conserved structural loops, loops A‐C. Although previous mutagenesis studies have identified His102 in Loop A as important for benzodiazepine modulation of GABA(A) receptors, the functional roles of many of the other conserved residues in loops A‐C remain incompletely understood. In this study, we made single mutations in loops A‐C of the benzodiazepine binding‐site across all six α subunits. We used whole‐cell patch clamp recording to measure the functional effects of these mutations on midazolam potentiation. The results showed that mutating the threonine in loop B and serine in loop C (Thr163 and S206 in human α1) did not abolish the receptors’ responsiveness to midazolam, as the α1(H102R) mutation did. The loop C mutations exhibited a novel array of α‐isoform specific effects on midazolam potentiation. The α3(S230I) and α5(S209I) mutations had the largest effect on midazolam potentiation, increasing the efficacy of midazolam. Novel benzodiazepines targeting loop C may represent a future direction for designing new drugs that specifically alter the activity of α3‐ and α5‐containing GABA(A) receptors.