Optopharmacology reveals a differential contribution of native GABAA receptors to dendritic and somatic inhibition using azogabazine

Abstract

γ-aminobutyric acid type-A receptors (GABA A Rs) are inhibitory ligand-gated ion channels in the brain that are crucial for controlling neuronal excitation. To explore their physiological roles in cellular and neural network activity, it is important to understand why specific GABA A R isoforms are distributed not only to various brain regions and cell types, but also to specific areas of the membrane in individual neurons. To address this aim we have developed a novel photosensitive compound, azogabazine, that targets and reversibly inhibits GABA A Rs. The receptor selectivity of the compound is based on the competitive antagonist, gabazine, and photosensitivity is conferred by a photoisomerisable azobenzene group. Azogabazine can exist in either cis or trans conformations that are controlled by UV and blue light respectively, to affect receptor inhibition. We report that the trans -isomer preferentially binds and inhibits GABA A R function, whilst promotion of the cis -isomer caused unbinding of azogabazine from GABA A Rs. Using cultured cerebellar granule cells, azogabazine in conjunction with UV light applied to defined membrane domains, revealed higher densities of GABA A Rs at somatic inhibitory synapses compared to those populating proximal dendritic zones, even though the latter displayed a higher number of synapses per unit area of membrane. Azogabazine also revealed more pronounced GABA-mediated inhibition of action potential firing in proximal dendrites compared to the soma. Overall, azogabazine is a valuable addition to the photochemical toolkit that can be used to interrogate GABA A R function and inhibition.

Photopharmacology of Ion Channels through the Light of the Computational Microscope

“…The trans -azogabazine docking poses revealed protein–ligand interactions similar to those observed in cryo-EM structures of other GABA R–antagonist complexes, explaining its antagonist activity. In addition, the trans -azogabazine docking poses were used to design site-directed mutagenesis experiments, which further confirmed the predicted binding mode [ 96 ].…”

“…Instead, the cis isomer is not able to place the 4-hydroxybenzylamine near the putative interacting residues, consistent with its lack of effect on channel function. Homology modeling and molecular docking were also used in a recent study [ 96 ] to rationalize the different binding preferences of azogabazine ( 14 ). This PCL is composed of azobenzene and gabazine, a known GABA R competitive antagonist binding to the orthosteric site.…”

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology

“…Subsequently, by exploiting a known single point mutation in the Shaker channel ion selectivity filter to increase its permeability to Na + [ 370 ], the depolarizing (D)-SPARK was engineered to allow control of depolarization and action potential generation in target neurons [ 371 ]. PTL are being continuously developed, and today, in addition to controlling ion channels, chemical optogenetics can be used to control ionotropic glutamate receptors [ 372 , 373 , 374 , 375 ], metabotropic glutamate receptors [ 376 ], GABA-A receptors [ 377 ], and acetylcholine receptors [ 378 , 379 ] (see also [ 354 ]).…”