Abstract
γ-aminobutyric acid type-A receptors (GABAARs) 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 GABAAR 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 GABAARs. 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 GABAAR function, whilst promotion of the cis-isomer caused unbinding of azogabazine from GABAARs. Using cultured cerebellar granule cells, azogabazine in conjunction with UV light applied to defined membrane domains, revealed higher densities of GABAARs 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 GABAAR function and inhibition.
Highlights
ABSTRACT γ-aminobutyric acid type-A receptors (GABAARs) are inhibitory ligand-gated ion channels in the brain that are crucial for controlling neuronal excitation
Using cultured cerebellar granule cells, azogabazine in conjunction with UV light applied to defined membrane domains, revealed higher densities of GABAARs 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
The fusion of gabazine and azobenzene involved sharing a common phenyl ring leading to the photoisomerisable gabazine analogue, azogabazine (Fig. 1A; Huckvale et al, 2016)
Summary
ABSTRACT γ-aminobutyric acid type-A receptors (GABAARs) are inhibitory ligand-gated ion channels in the brain that are crucial for controlling neuronal excitation. To develop a compound that can reversibly and repeatedly inhibit targeted GABAARs we have modified the antagonist gabazine to enable light-activated cis:trans conformational isomerization (Huckvale et al, 2016) This new photochemical molecule, called azogabazine (AGZ), has been used to explore the mechanism of antagonist-based isomerization, and how this may be used to dissect synaptic inhibition by targeting the proximal-dendritic and somatic regions of neurons using membrane delimited-photoisomerisation. This revealed differential contributions of GABAARs located in discrete cell membrane sub-domains to inhibition of cerebellar granule neurons
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