Light-induced regulation of ligand-gated channel activity

Abstract

The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field.

Linked articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Figure 1

Optical switches for modulating the activity of nAChRs. (A) Light‐induced conformations of azobenzene. (B) Chemical structures of the first azobenzene‐based PCLs for regulating the function of nAChRs (modified from Lester et al., 1980). (C) PTLs for photochemical control of neuronal AChRs. (C, panel a) Modular organization of MAACh in trans configuration. (C, panel b) Scheme of a tethered agonist action. At illumination, with visible light (500 nm) or in darkness, the compound is in its trans‐configuration and not capable of activating heteropentameric nAChRs (upper part). Under UV light (380 nm), the tethered agonist is converted into its cis‐configuration and thus activates receptors causing the channels to open (bottom part). (C, panel c) Photoactivation of the α3β4E61C mutant receptors by tethered MAACh in Xenopus oocyte. Illumination at 380 nm (violet line) triggers ionic current and, at 500 nm (green line), shuts it off. For comparison, the right trace shows the response to ACh 100 μM. (C, panel d) Photoinhibition of the current induced by 300 μM ACh (green line) and the effect when tethered to the α3β4E61C mutant receptor antagonist MAHoCh at 380 nm illumination (violet line; modified from Tochitsky et al., 2012). (D) Photoswitchable PCL agonist for neuronal α7 nAChRs. (D, panel a) Chemical structure of the AzoCholine. (D, panel b) Light‐dependent effect of BisQ or AzoCholine on α7/Gly receptors chimera expressed in HEK293T cells. Note that illumination at 440 nm triggered a large inward current (bottom trace) while BisQ was not effective (top trace). (D, panel c) Effect of BisQ or AzoCholine on neuromuscular nAChR (α1/β1/δ/ε) expressed in HEK293T cells. Note that on this receptor, AzoCholine is not active, in contrast to BisQ (modified from Damijonaitis et al., 2015a).

Figure 2

Optical switches for modulating the activity of glutamate receptors. (A) Modular design of azobenzene–glutamate photoswitches. (A, panels a, b) PTLs in trans configuration for modulation of ionotropic glutamate receptor. They are composed of three parts: maleimide–azobenzene–glutamate. In (A, panel a), for clarity, different components of the synthetic photoswitcher are highlighted and labelled. For MAG₃₈₀ (A, panel a), the most efficient isomerization from trans to cis configuration is triggered by illumination at 380 nm (Volgraf et al., 2006), while for L‐ MAG0₄₆₀ (A, panel b), this transition occurs at visible light with optimal wavelength 460 nm (Kienzler et al., 2013). (A, panel c) PTL agonist for native affinity labelling via lysines. There was no need to introduce cysteine by mutagenesis (from Izquierdo‐Serra et al., 2016). (A, panel d) PCL version of azobenzene–glutamate photoswitcher, which reversibly interacts with the glycine receptor (modified from Volgraf et al., 2007). (B, panel a) The ribbon structure of apo‐iGluR2 together with the ball‐and‐stick structure of MAG attached to cysteine at L439C (yellow) in the extended (trans) and unbound conformation (modified from Gorostiza et al., 2007). (B, panel b) A neuron transfected with iGluR6 (L439C) and labelled with MAG is illuminated at 380 nm for 500 ms, yielding reproducible depolarization that triggers trains of action potentials. Illumination at 500 nm turns the response off and permits repolarization. (Modified from Szobota et al., 2007). (C) The photo‐induced activation of LiGluR with ‘red‐shifted’ covalently tethered MAG₄₆₀. (C, panel a) Patch‐clamp recording from HEK 293 cells expressing GluK2 (439C). Illumination at 500 nm induces the generation of inward currents, while in the dark MAG₄₆₀ relaxes back to a trans configuration, resulting in the closing of the channels (modified from Kienzler et al., 2013). (C, panel a) The effect of blue light illumination (blue bar) on activity of cultured hippocampal neuron expressing a mutant of GluK2 with a cysteine substitution (L439C). Current‐clamp recording (modified from Levitz et al., 2016).

Figure 3

Optical switches that modulate the activity of ionotropic GABA receptors. (A) Examples of chemical structures of some PCL (A, panels a, b) and PTL (A, panel c) PCLs of GABA receptors. (B) Ion current induced on Xenopus oocyte expressing α1β2γ2 GABA_A receptors. (B, panel a) Left trace; current induced by 3 μM GABA; right trace: co‐application of 3 μM GABA and 1 μM MPC088 at visible light and during illumination with UV light. (B, panel b) Ion current induced by application of 15 μM MPC088 at visible light and during repetitive illumination of the oocyte with UV light. Note that UV illumination attenuates the responses, while at visible light, the currents slowly recover. (B, panel c) Whole‐cell recording from the mouse brain slice. Effect of MPC088 photoactivation on GABA‐evoked currents in cerebellar Purkinje neuron. Cells were exposed to multiple UV/blue light flashes during the application of GABA and MPC088 (indicated above the trace; modified from Yue et al., 2012). (C, panel a) Scheme of photoswitchable PTL antagonist MAM‐6 action; after it is conjugated to the GABA receptor, it reversibly isomerizes between the cis‐ and trans‐ states. In the cis‐configuration (UV illumination), it is not active, while at illumination with visible light, it isomerizes to its trans‐configuration and prevents GABA binding and the subsequent opening of the channels. (C, panel a) Photoregulation of GABA‐induced currents by the tethered MAM‐6 on cells expressing the mutant S68C of α1 GABA receptor subunits (modified from Lin et al., 2014) (D) Differential photo‐control of inhibitory postsynaptic currents in cerebellar molecular layer interneuron (top traces) and a Golgi cell (bottom traces) of the mouse expressing the α1‐GABA_A receptor with a single point mutation (T125C) and treated with PCL compound PAG‐1C. Note that on a Golgi cell, the currents are not modulated by light, suggesting the absence of α1‐GABA_A receptors on these cells (modified from Lin et al., 2015).