Molecular determinants underlying DS2 activity at δ-containing GABA_A receptors

Abstract

Delta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by δ subunit-containing GABA_A receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive.

Using a combination of computational modeling, site-directed mutagenesis and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α₄β₁δ receptors: an α₄⁽⁺⁾δ^(-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ₂ receptors, and two sites in the transmembrane domain (TMD); one in the α₄⁽⁺⁾β₁^(-) and one in the α₄^(-)β₁⁽⁺⁾ interface, with the α₄^(-)β₁⁽⁺⁾ site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α₄⁽⁺⁾β₁^(-) interface, either α₄(S303L) of the α₄⁽⁺⁾-side or β₁(I289Q) of the β₁^(-)-side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patchclamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points towards a functional selectivity of DS2 and its analogs via a surprisingly well-conserved binding pocket in the TMD.