Functional β1-selectivity of novel pyrazoloquinolinones at GABAA receptors revealed by electrophysiology, mutagenesis and docking.

Gamma-aminobutyric acid type A (GABAA) receptors are ligand-gated anion channels, encoded by nineteen subunit genes in mammals. Recent cryo-electron microscopy studies of native human receptors from brain cortex revealed that β1 subunits contribute to diverse assemblies, including non-canonical arrangements of subunits. β subunits contribute to both GABA binding sites and allosteric sites targeted by general anesthetics. However, studying β1-containing receptors remains hampered by the absence of selective ligands. Here, we present novel pyrazoloquinolinone (PQ) ligands that act as functionally selective allosteric modulators and propose a structural hypothesis for their binding site. Two-microelectrode voltage clamp recordings in Xenopus laevis oocytes expressing recombinant GABAA receptors were used to generate concentration-response curves, asses functional β1-selectivity and probe the impact of a mutation in the putative binding site. Four PQ derivatives significantly enhanced GABA-induced currents at α1β1 receptors but showed markedly reduced efficacy at α1β3 receptors, demonstrating functional β1-selectivity. Incorporation of the γ2 subunit reduced or abolished this modulation, and mutational analysis identified β1R41 as key determinant of activity. The emerging structure-activity relationships define a distinct interaction profile and provide quantitative evidence for β1-selectivity. Together, our results pave the way for further design of binding selective ligands. Future applications of β1-selective compounds include their use as research tools for example in autoradiography or functional mapping of receptor populations, and perspectives as diagnostic and therapeutic agents in a variety of neuropsychiatric conditions, as the subunit encoding GABRB1 gene has been implicated in epileptic encephalopathy and is under investigation for a potential role in addictive disorders.