Falk-Petersen, C. B. | bioRxiv Science

bioRxiv2021

Molecular determinants underlying DS2 activity at δ-containing GABAA receptors

Delta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by {delta} subunit-containing GABAA 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 4{beta}1{delta} receptors: an 4(+){delta}(-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in {beta}{gamma}2 receptors, and two sites in the transmembrane domain (TMD); one in the 4(+){beta}1(-) and one in the 4(-){beta}1(+) interface, with the 4(-){beta}1(+) 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 4(+){beta}1(-) interface, either 4(S303L) of the 4(+)-side or {beta}1(I289Q) of the {beta}1(-)-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 {delta}-containing receptors is caused by a direct interaction with the {delta}-subunit, and instead points towards a functional selectivity of DS2 and its analogs via a surprisingly well-conserved binding pocket in the TMD. Significance statement{delta}-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition. Yet, no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the know compound DS2, the ground is laid for design of ligands that selectively target {delta}-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and, ultimately, for rational development of novel drugs.

pharmacology and toxicology↗

bioRxiv2018

Kinase activity simultaneously determines the constitutive and the orthosteric gating in α4β1/3δ GABAA receptors in hippocampal granule cells

A subset of the GABAA receptors expressed in recombinant systems and neurons is known to exhibit both constitutive- and agonist-induced gating. Two such receptors are the {delta}-subunit containing GABAA receptors 4{beta}1{delta} and 4{beta}3{delta}, which are expressed in adult rodent hippocampal dentate gyrus granule cells (DGGCs). Here we show that the GABAA receptor mediated tonic current recorded in the presence of tetrodotoxin in adult rodent DGGCs is almost exclusively mediated by constitutively active {delta}-subunit containing GABAA receptors and that the constitutive current is absent in recordings at 24 {degrees}C or in recordings at 34 {degrees}C including an intracellular inhibitor of protein kinase C. These factors simultaneously govern the efficacy of an orthosteric agonist at 4{beta}1/3{delta} receptors, Thio-THIP, in a reciprocal manner. In the absence of constitutive receptor activity, the efficacy of Thio-THIP was increased approximately four-fold relative to recording conditions that favors constitutive activity. Further, only under conditions of an absent constitutive current, the classified neutral antagonist gabazine (GBZ) alone, induced a tonic current in DGGCs (EC50 2.1 M). This effect of GBZ was not seen in recording conditions of high constitutive activity, was inhibited by picrotoxin (PTX), potentiated by DS2, completely absent in {delta}-/- mice and reduced in {beta}1-/- mice, but could not be replicated in human 4{beta}1/3{delta} receptors expressed recombinantly in HEK cells. We hypothesize that specific intracellular components in neurons interact with receptors to determine constitutive gating and receptor responsiveness to orthosteric ligands.\n\nSignificance statementThe presented data highlight how recording conditions for whole cell patch clamp analysis of 4{beta}1/3{delta} GABAA receptors can mask important pharmacological effects. Specifically, orthosteric agonists appear with reduced efficacy, and other ligands, here exemplified with the well-known antagonist GBZ, are misinterpreted as being inactive/neutral, although they could have effect in constitutively silent receptors. Unmasking of potential hidden effects are easily done using recording conditions of reduced kinase activity in a relevant neuronal context. It follows that in pathologies with changes in phosphorylation level of {delta}-subunit containing GABAA receptors, the efficacy of an agonist of these receptors, measured by whole-cell recordings in vitro, will not match the efficacy of the same agonist in an unperturbed neuron in vivo.

neuroscience↗