A photoswitchable GABA receptor channel blocker

Abstract

Background and Purpose Anion‐selective Cys‐loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride‐selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light‐controllable modulator of GABA receptors, we constructed azobenzene‐nitrazepam (Azo‐NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group. Experimental Approach The experiments were carried out on cultured cells expressing Cys‐loop receptors of known subunit composition and in brain slices using patch‐clamp. Site‐directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo‐NZ1. Key Results At visible light, being in trans‐configuration, Azo‐NZ1 blocked heteromeric α1/β2/γ2 GABAA receptors, ρ2 GABAA (GABAC), and α2 glycine receptors, whereas switching the compound into cis‐state by UV illumination restored the activity. Azo‐NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans‐configuration, Azo‐NZ1 blocks the Cl‐selective ion pore of GABA receptors interacting mainly with the 2′ level of the TM2 region. Conclusions and Implications Azo‐NZ1 is a soluble light‐driven Cl‐channel blocker, which allows photo‐modulation of the activity induced by anion‐selective Cys‐loop receptors. Azo‐NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination.

Optical Control of GABAA Receptors with a Fulgimide‐Based Potentiator

“…A powerful alternative is using reversible chemical photoswitches to harness endogenous anion‐conducting receptor‐channels like GABA and glycine receptors (GABA A R, GlyR), which mediate inhibitory neurotransmission in the mammalian central nervous system . Although some GABA A R photoswitches have been reported based on azobenzene, this photochromic group displays several shortcomings: it provides incomplete photoconversion due to a substantial overlap of the absorption maxima of cis and trans isomers, and can alter the pharmacophore activity. Indeed, in all azobenzene derivatives of benzodiazepines (allosteric potentiators of GABA A R) this characteristic property is abolished, as found in the 7‐amino site of nitrazepam, which is reportedly tolerant of other substitutions .…”

“…To characterize Fulgazepam in vitro, patch clamp experiments were performed on cells transiently expressing α 1 β 2 γ 2 subunits of the GABA A receptor. This receptor possesses the canonical benzodiazepine allosteric site and its EC 50 for GABA is about 8 μ m . The effects of the fulgimide‐based benzodiazepine derivatives 3 and 4 on the receptor's function were studied upon co‐application of 0.5 μ m GABA, that is, a concentration below the EC 50 (close to EC 3 ) that allows to observe allosteric potentiation of GABA A R‐mediated currents …”

“…We have recently developed an azobenzene‐nitrazepam based compound (Azo‐NZ1) that allows photo‐modulation of GABA A receptors . In trans ‐configuration, this compound selectively interacts with the chloride‐permeable channel causing inhibition of GABA‐induced currents, while UV‐induced transition to the cis ‐state results in channel unblocking and restoring the current amplitude.…”

Fulgazepam: A Fulgimide-Based Potentiator of GABAA Receptors

“…This receptor possesses the canonical benzodiazepine allosteric site and its EC50 for GABA is about 8 µM. [54] The effects of the fulgimide-based benzodiazepine derivatives 3 and 4 on the receptor's function were studied upon co-application of 0.5 µM GABA, i.e. the concentration, which is below the EC50 (close to EC3) and allows to observe allosteric potentiation of GABAAR-mediated currents.…”

Subunit-Specific Photocontrol of Glycine Receptors by Azobenzene-Nitrazepam Photoswitcher

“…It has been recently shown that Azo-NZ1, the azobenzene derivative of nitrazepam, blocks in a UV-dependent manner the ion channel pore of GABA A alpha1/beta2/gamma2 and GABA C rho2 receptors and controls GABAergic currents (Maleeva et al, 2019). These experiments also suggested that Azo-NZ1 is capable to modulate the activity of another subtype of Cys-loop receptors -alpha2 GlyR.…”