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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Oct 6;175(11):1892–1902. doi: 10.1111/bph.14022

Light‐induced regulation of ligand‐gated channel activity

Piotr Bregestovski 1,2,✉, Galyna Maleeva 1, Pau Gorostiza 3,4,5
PMCID: PMC5979632  PMID: 28859250

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

Abbreviations

AP2

azobenzene‐propofol

ATA

azobenzene‐tetrazolyl‐AMPA

LiGluR

light‐activated ionotropic glutamate receptor

MAB

maleimide‐azobenzene‐4‐hydroxybenzylamine

MAG

maleimide‐azobenzene‐glutamate

MAM

maleimide‐azobenzene‐muscimol

nAChR

nicotinic ACh receptor

PCL

photochromic ligand

PTL

photochromic tethered ligands

TCPs

targeted covalent photoswitches

Introduction

Starting with Antoni van Leeuwenhoek and following the highly important contributions of Camillo Golgi and Santiago Ramón y Cajal, optical methods have been embedded at the heart of scientific research and are still widely used to elucidate the morphology and function of different cell types, as well as to determine the principles of the organization of biological organisms. The rapid progress in molecular biology and fluorescent microscopy in combination with the use of genetically‐encoded sensors has significantly expanded the possibilities of optical studies. The development of methods for the specific integration of proteins in certain cell types, as well as the detection of light‐sensitive proteins, has stimulated the explosion of knowledge of the remote control and specificity.

As a result, in recent years, new fields, namely, optogenetics, optopharmacology/photopharmacology and optogenetic pharmacology, have been developed (Fenno et al., 2011; Broichhagen et al., 2015; Repina et al., 2017).

The origin of optogenetics has been stimulated by the cloning of the first light‐gated cation‐selective membrane channel, channelrhodopsin (Nagel et al., 2003) and observations that its expression in cells results in the ability of these cells to be activated by light, which results in changes in membrane potential, the generation of ion currents and increased firing of the cells (Nagel et al., 2003; Boyden et al., 2005). Moreover, it was found that integrating photosensitive proteins into neurons of multicellular organisms allows the application of light to be used to change their behaviour (Lima and Miesenböck, 2005). Thus, it has become evident that bacterial light‐sensitive proteins represent rather simple and easy‐to‐use tools for the rapid control of cell excitability and function of neural networks. New light‐sensitive proteins and their derivatives have been embedded in cells of different species of animals from worms and insects to monkeys (Diester et al., 2011; Fenno et al., 2011; Han et al., 2011; Gerits et al., 2012; Welberg, 2012) and human pluripotent cells (Busskamp et al., 2010; Steinbeck et al., 2015).

By controlling the activity of cells with the help of light, one can investigate their function (Tye and Deisseroth, 2012), measure the concentration of ions (Bregestovski et al., 2009), ATP (Berg et al., 2009; Imamura et al., 2009) and other cellular components (Bilan et al., 2013; Marvin et al., 2013), control the behaviour of organisms (Covington et al., 2010; Haubensak et al., 2010; Miesenböck, 2011) and find novel ways of treating certain diseases (Laprell et al., 2015; Rossi et al., 2015). Optogenetic approaches have been used in many models with a medical orientation, including the study of stress (Covington et al., 2010), schizophrenia, memory disorders, drug addiction, psychiatry and motor functions (Rossi et al., 2015); vision, pain, functional recovery after stroke and epilepsy (Tønnesen et al., 2009; Gaub et al., 2014; Wagner et al., 2015). However, one of the critical limitations of optogenetics is the necessity to integrate foreign genes into organisms using viral gene therapy or the development of transgenic animals.

In parallel with optogenetics, in recent years, photopharmacology or optopharmacology – an approach based on the creation of chemical compounds capable of controlling the functions of biological molecules with the help of chemical photosensitive switches – has greatly developed (Kramer et al., 2013). Several classes of photochromic pharmacological compounds have already been successfully used to selectively modulate the activity of various proteins, including enzymes (Harvey and Abell, 2001), receptor‐driven (Szobota et al., 2007; Tochitsky et al., 2012) and potential‐dependent ion channels (Banghart et al., 2004; 2009; Fortin et al., 2008; 2011).

While the use of some photoswitchable compounds requires the mutation of target proteins, a number of improved soluble compounds with a high specificity of action have been developed that do not require genetic manipulations. It has emerged that synthetic light‐controlled compounds capable of enhancing or inhibiting the activity of key cellular proteins are powerful tools for the non‐invasive control of cellular activity, function of organs and the behaviour of living organisms.

In this mini‐review, we will focus on the control of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=697&familyType=IC by using light‐sensitive molecules or photoswitches. In particular, we will introduce the early developments in the photocontrol of http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=76 on the neuromuscular junction and more recent progress with the main excitatory and inhibitory receptor‐channels in the vertebrate nervous system (http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=75 and http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=72 receptors, respectively). More comprehensive reviews, including those on voltage‐gated, transient receptor potential and trimeric receptor ion channels, have been recently published elsewhere (Bautista‐Barrufet et al., 2014; McKenzie et al., 2015).

In general, photoswitches can be divided into two main classes: (i) those acting as soluble photochromic ligands (PCL); and (ii) those that are covalently tethered to the target protein (PTL, photochromic‐tethered ligands). Each class has its own advantages and limitations.

PCLs are convenient and simple to use in endogenous receptors and do not require molecular modification (e.g. mutagenesis). However, it is difficult to obtain highly specific PCLs, as many receptor proteins show high similarity in ligand binding sites, which are conserved in most cases.

The PTL strategy allows the activity of voltage‐gated or receptor‐operated ion channels to be controlled, due to irreversible tethering of PTLs to the proteins, often targeted at cysteine residues, that either occur naturally or are genetically introduced (Lester et al., 1980; Gorostiza and Isacoff, 2007; 2008; Kramer et al., 2013). In general, this strategy requires both (i) chemical synthesis of PTL compounds and (ii) mutagenesis of the target protein to identify a suitable tethering site for optimal photoswitching. It offers the advantage of activating or inhibiting only this specific receptor or ion channel mutant. On the other side, because of the need, in some cases, to mutate target proteins, this poses limitations and difficulties when used in experimental models in vivo, and for testing on humans.

Photoswitches (both PCLs and PTLs) are chemically synthesized molecules containing at least two components: a ligand molecule (agonist, antagonist or ion channel blocker) and a photoisomerizable group. PTLs, in addition, have a reactive group for irreversible tethering to the target protein (Figure 1).

Figure 1.

Figure 1

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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).

The synthetic photoswitch that is most extensively used for channel applications is azobenzene (Figure 1A), a molecule, which undergoes cis‐trans‐isomerization around the central double N═N bond (see rev Gorostiza and Isacoff, 2007; McKenzie et al., 2015). In the dark or under visible light, azobenzene is in an extended trans configuration. Irradiation with near‐UV light (360–380 nm) induces a change from the trans to the cis configuration, which shortens the molecule by about 0.6 nm. Visible light switches the azobenzene back to the trans form (Figure 1A). Isomerization of azobenzene occurs in picoseconds upon absorption of a UV photon (Bortolus and Monti, 1979), and this permits high‐speed switching of many azobenzene‐based molecules using bright light. Thermal back‐relaxation lifetimes range between milliseconds and days and can be adjusted by synthetic design according to application requirements (Velema et al., 2014).

Control of the nicotinic acetylcholine receptor using light

The nicotinic ACh receptor (nAChR) was the first light‐modulated receptor‐operated channel. Almost 50 years ago, Deal et al. (1969) introduced azobenzene‐based PCLs for regulating the activity of the nAChR. Firstly, the team demonstrated the ability of azobenzene‐based compounds to act as photochromic regulators of enzymatic activity of chymotrypsin (Kaufman et al., 1968) and AChE (Bieth et al., 1969) and then extended this idea to the light‐induced regulation of the excitability of the electroplax preparation of Electrophorus electricus. They used N‐p‐phenylazophenyl‐N‐phenylcarbamylcholine chloride and p‐phenyl‐azophenyl‐trimethylammonium chloride as light‐sensitive antagonists of the ACh receptor (Deal et al., 1969). This pioneer study was the first demonstration of the use of photochromic compounds to regulate the activity of cys‐loop receptor channels.

Over the years, both classes of photoswitches (PCL and PTL) for modulating the function of nAChRs have been developed. They represent Bis‐Q, a compound whose two ligand moieties can bind reversibly to the receptor, and tethered QBr, which can be covalently linked to the native sulfhydryl groups in the vicinity of ACh binding site (Figure 1B). Irreversible targeting could be achieved after the treatment of receptors with dithiothreitol, a reducing agent that causes a reduction of the disulfide (S─S) bonds and exposes free thiols to the reaction (Karlin, 1969; Bregestovski et al., 1977). These compounds, called ‘tethered’ agonists, were successfully used to induce light‐flash relaxations and to analyse rate‐limiting steps governing the opening and closing of channels (Bartels et al., 1971; Bartels‐Bernal et al., 1976; Lester et al., 1979, 1980; Chabala et al., 1986).

These developments were to a large extent empirical and cumulative, but determination of the amino acid sequences and atomic structures of the channel proteins brought about the possibility of rationally designing photochromic compounds to regulate the activity of ligand‐ and voltage‐gated channels, and a rebirth of the field in the twenty first century (Banghart et al., 2004; Volgraf et al., 2006; Szobota et al., 2007; Gorostiza et al., 2007; Stein et al., 2012; Yue et al., 2012; Damijonaitis et al., 2015a, b).

Recently, new generations of PCLs and PTLs for effective light‐dependent modulation of nAChRs were proposed. Based on the X‐ray structure of an http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=294 binding protein in complex with http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=298 (Celie et al., 2004) and distance measurements in the protein structures, in the β‐subunit of nAChR, several positions that face the ligand binding site have been identified as a potential region for the attachment of agonists and antagonists. This has allowed site‐directed insertions of cysteines to be performed and the synthesis of appropriate PTLs.

After mutated nAChRs were expressed in Xenopus oocytes of and labelled with the PTL agonist [maleimide–azobenzene–ACh (MAACh)] or antagonist [maleimide‐azobenzene‐homocholine (MAHoCh)], illumination with a 380 nm light produced either an inward current that could be reversed with 500 nm light (labelling with MAACh), or ACh‐induced currents could be inhibited by this light by labelling with MAHoCh (Figure 1C). These PTL compounds enabled heteromeric neuronal nAChRs to be activated or inhibited with UV light but respond normally to ACh in the dark, which is important for a more profound analysis of their physiological and pathological cholinergic functions (Tochitsky et al., 2012).

Recently, the team of D. Trauner have reported a photoswitchable agonist for neuronal http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=468, AzoCholine (Damijonaitis et al., 2015a). In an α7 nACh/glycine receptor chimera heterologously expressed in HEK293T cells, this compound was not effective when exposed to UV light; however, it caused a large current when illuminated with visible light, that is, at transition to trans configuration (Figure 1D). AzoCholine thus activates α7 receptors in the dark, but on the other hand, it displays subtype selectivity with regard to the muscular nAChR. Importantly, AzoCholine is a PCL compound, that is, its application does not need molecular modification of the α7 nAChRs. AzoCholine effectively modulated the neuronal activity of rat sensory neurons from dorsal root ganglia in mouse hippocampal brain slices, and it was able to modulate, in a light‐dependent manner, the swimming behaviour of C. elegans (Damijonaitis et al., 2015a). This demonstrates that the main advantage of AzoCholine compared to other PCLs, is the ease with which it can be used for light‐dependent control of cellular processes in vitro and in vivo.

This area of research is now developing extremely rapidly and in various directions. Below, we will discuss just some of the studies, concentrating on the two main functional classes of ionotropic receptors that determine synaptic excitation and inhibition in the nervous system of vertebrates.

Photochromic modulators of glutamate receptors

Glutamate receptors provide the main excitatory drive in the mammalian nervous system and are involved in a large variety of physiological processes, including brain development, synaptic plasticity, memory formation, pain, excitotoxicity and neurodegenerative diseases (Gonzalez et al., 2015; Zhuo, 2017). Disorders of glutamatergic transmission lead to imbalances in inhibition‐excitation and have dramatic consequences for both cellular and network functions. These receptors are among the primary targets for the development of photopharmacological regulators.

The first photoswitch for glutamate receptors was engineered 10 years ago (Volgraf et al., 2006; Gorostiza et al., 2007) and termed maleimide‐azobenzene‐glutamate (MAG) to highlight its components: a cysteine‐reactive maleimide group, an azobenzene photoswitch and a glutamate ligand (Figure 2A, panel a). Maleimide allowed the compound to be tethered to the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=451 after introducing a cysteine substitution (L439C), close to the glutamate‐binding site (Figure 2B, panel a). This type of receptor was called a LiGluR – light‐activated ionotropic glutamate receptor. Illumination with 380 nm light induced the transition of MAG to its cis state, in which the glutamate head was bound to the agonist‐binding site with subsequent activation (opening) of the ion channel. Back isomerization of MAG and receptor deactivation were triggered by exposure to 500 nm light (Volgraf et al., 2006). Light pulses reliably induced the depolarization and firing of neurons due to the activation of GluK2 channels by MAG (Figure 1B, panel b; Szobota et al., 2007).

Figure 2.

Figure 2

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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).

Later, the same group proposed the first nontethered photochromic agonist of the inhibitory glutamate receptors that could modulate the function of wild‐type receptors, glutamate‐azobenzene (Figure 2A, panel d). This compound, which represents the PCL series, was based on using a potent and selective agonist of the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=450 chemically conjugated to an azobenzene (Volgraf et al., 2007). The photoswitchable ligand was controlled by the same wavelengths as MAG, and its activity was competitively blocked by the non‐NMDA receptor antagonist DNQX. In contrast to MAG, this compound activated kainate receptors in the trans‐state and lost its activity in the cis‐configuration induced by UV light. This PCL successfully caused light‐induced modulation of depolarization in cultured hippocampal neurons from wild‐type rats, demonstrating that the cysteine substitution in the target receptor is not required for its action (Volgraf et al., 2007).

The results of these pioneering works were further elaborated in a number of subsequent studies, demonstrating that photoregulation of glutamate receptors represents an efficient tool for controlling glutamatergic neurotransmission. One of the aspects to be optimized was the photoswitch action spectrum. The requirement of UV light for azobenzene isomerization is not ideal for biological systems because (i) prolonged UV exposure can be damaging and (ii) UV light poorly penetrates mammalian tissue. To overcome this problem, a light‐sensitive azobenzene‐based glutamate receptor ligand with about 100 nm redshift of the absorption was synthesized (Kienzler et al., 2013). The compound, called MAG460 (Figure 1A, panel b), can be induced to switch to its cis‐configuration by visible light (460 nm) and rapidly returns to the trans‐state when subjected to thermal relaxation in the dark (Figure 2). Whole‐cell patch‐clamp recording from HEK293 cells expressing GluK2(439C) and incubated with the red‐shifted L‐MAG0460 showed that illumination with blue light induced a large inward current (Figure 2C, panel a). In the dark, the currents were observed to recover due to the closing of the channels after MAG460 had returned to its trans configuration. Other MAG variants, with slower kinetics, can be activated with red light (625 nm) (Rullo et al., 2014).

Recently, LiGluRs have been expressed in the visual cortex of mice using an adeno‐associated virus (AAV) under the control of a specific promoter. In conjunction with fibre‐based optogenetic technologies, it has been shown that MAG0460 can activate LiGluRs in cultured hippocampal neurons (Figure 2C, panel b) and, in in vivo conditions, neuronal cell firing in the mouse cortex can be increased by exposure to blue light (Levitz et al., 2016). These experiments have demonstrated that the LiGluR‐MAG technique is compatible with existing fibre‐based in vivo light control technologies and can be used to manipulate the activity of the neuronal circuitry.

Similar blue‐shifted MAG derivatives were developed for the purpose of enhancing two‐proton activation of the azobenzene switch using pulsed infrared light (Izquierdo‐Serra et al., 2014; Gascón‐Moya et al., 2015), which penetrates deeper into tissue and enables focal activation in neurons and astrocytes. Two photon activation and digital holography were further used to shape stimulation patterns in three dimensions for the purpose of studying neural circuits (Carroll et al., 2015).

The development of MAG derivatives isomerized by visible light also expanded the application of LiGluRs in vision restoration research (Kienzler et al., 2013). The first attempt at using light‐sensitive glutamate ligands for vision restoration was performed with UV‐modulated MAG. The gene encoding for cysteine substituted into the GluK2 subunit of the glutamate receptor (LiGluR) was delivered to retinal ganglionic cells by intravitreal injection of AAV, and the photoswitchable‐tethered ligand MAG was delivered in a subsequent intravitreal injection. This resulted in a restoration of the light responses of blind mice with retina degeneration (Caporale et al., 2011).

However, as mentioned above, the use of UV light raises some problems, particularly in the case of retina. The second generation of red‐shifted LiGluR‐MAG0460 has been shown to be much more promising. Upon administration of MAG0460, light‐evoked responses in retinal ganglion cells as well as in ON‐bipolar cells were recorded. Moreover, visual‐guided behaviour of animals was demonstrated in the functional tests in blind mouse and dog models (Gaub et al., 2014).

The structural data combined with mutagenesis and electrophysiological observations (Sobolevsky, 2015) greatly facilitated the design of effective photoswitches for AMPA, kainate and NMDA receptors. Photoswitchable activators of AMPA receptors were developed on the basis of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4131 with azobenzene substitution and were called azobenzene‐tetrazolyl‐AMPAs (ATA). They were shown to be potent AMPA‐activators in the trans‐state and could be used to control neuronal activity in acute cortical brain slices (Stawski et al., 2012; Reiner et al., 2015).

MAG‐based ligands also enabled the photoregulation of NMDA‐selective glutamate receptors. The previously described method of cysteine substitution yielded light‐activated http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=456, light‐activated http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=457, light‐antagonized GluN2A and light‐antagonized http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=455 subunits of the NMDA receptor (Berlin et al., 2016). This model of light‐controlled NMDA receptor subunits provides precise, fast and reversible remote control of specific receptor subtypes in localized areas, modulation of excitatory synaptic currents, long‐term plasticity and spine‐specific regulation of intracellular calcium transients.

Variants in the reactive group of MAG derivatives have also been explored, with the aim of achieving covalent conjugation (PTL) that does not require the introduction of cysteine residues by mutagenesis, thus targeting endogenous receptors (Figure 2A, panel c; Izquierdo‐Serra et al., 2016). A modular library of photoswitchable ligands and reactive groups was optimized for GluK1 and allowed the identification of effective photoswitches that covalently conjugated to a lysine residue in the receptor following an affinity labelling process. Thus, they can be termed photoswitchable affinity labels (Harvey and Trauner, 2008) or targeted covalent photoswitches (TCPs) in analogy with targeted covalent drugs, an important class of medicines that include aspirin, penicillin and omeprazole. These compounds activate GluK1 under UV light and deactivate it under 500 nm illumination, providing photocontrol of untransfected neurons and restoration of the photosensitivity of degenerated retina.

The currently available photochromic modulators of glutamate receptors offer a wide choice of pharmacological function (agonist, antagonist), selectivity (http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4231 AMPA, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4268) and optical properties (from violet to red to IR stimulation using multiphoton processes and different relaxation lifetimes in the dark). In addition, it is possible to take advantage of genetic targeting using cysteine‐conjugated MAG derivatives or aim at endogenous receptors using either freely diffusible PCLs or lysine‐targeted photoswitches that are conjugated by affinity. Overall, the photoswitch toolbox is well equipped to provide a systematic investigation of glutamatergic neurotransmission in the mammalian brain.

Light‐induced modulation of GABA receptors

Since http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1067 provides the main inhibitory neurotransmission in the CNS of vertebrates, the search for specific photoswitchable regulators of GABA receptor function constitutes a very important task. Due to the efforts of several teams, a diverse range of optically switched ligands of GABAA receptors has been developed, including PTLs and PCLs, activators, allosteric potentiators and antagonists.

One of the first compounds that served as a basis for development of light‐sensitive potentiators of the GABAA receptor was http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5464. This lipophilic anaesthetic has been shown to act as potentiator of GABA‐induced currents (Sieghart, 1995). A propofol/azobenzene‐based photo‐isomerizable soluble ligand MPC088 (Figure 3A, panel a) was developed by the team of David Pepperberg (Yue et al., 2012). Using α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes, it was shown that in the trans‐form, this freely diffusible PCL type compound effectively potentiated GABA‐induced currents at a concentration of 1 μM, while at higher concentrations, it directly activates the receptors (Figure 3B, panel a). In the cis‐form generated by UV‐illumination (365 nm), the compound had little effect on the amplitude of GABA‐induced currents. Moreover, in cerebellar brain slices, MPC088 co‐applied with GABA caused bidirectional photomodulation of the Purkinje cell membrane current (Figure 3B, panel b) and changes in spike‐firing rate (Yue et al., 2012). The results of this study suggest that MPC088 interacts with GABA receptors at the same β subunit site as propofol, but the efficiency of this interaction is higher than that with propofol.

Figure 3.

Figure 3

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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 GABAA 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‐GABAA 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‐GABAA receptors on these cells (modified from Lin et al., 2015).

Another chemically synthesized azo‐propofol compound, AP2, which contains an azobenzene group at the para‐position of phenol, has been reported (Figure 3A, panel b; Stein et al., 2012). In its trans‐configuration, AP2 potentiated GABA‐induced currents with an EC50 in the μmol range, while irradiation with UV light, which shifted the compound into its cis‐configuration, prevented the development of this potentiation effect. Propofol‐based AP2 was demonstrated to be active in Xenopus oocytes, HEK cells and in an animal model – Xenopus laevis tadpoles, where it caused light‐dependent anaesthesia. Future studies in other experimental models should demonstrate the usefulness of AP2 as a light‐dependent anaesthetic and modulator of GABAergic activity in the brain. However, it is possible that trans‐MPC088 and AP2 also modulate the function of non‐GABAA receptor ion channels or other proteins of neural tissues.

More recently, two effective inhibitory PTLs were tethered to the mutant GABA receptor (LiGABAAR) that contains a cysteine‐substituted α1 subunit (T125C) (Lin et al., 2014). One of the compounds [maleimide‐azobenzene‐muscimol (MAM) 6] consists of http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4259 (element responsible for the specific interaction with GABA‐binding site) combined with an azobenzene photoswitch conjugated to maleimide. Effective allocation of the compound to the active site was achieved using a 6‐carbon spacer between the muscimol pharmacophore and the azobenzene group (Figure 3A, panel c).

Although muscimol is an agonist for ionotropic GABA receptors (Johnston, 1996; Krogsgaard‐Larsen et al., 1997), the MAM‐6 acted as a photoswitchable antagonist, able to bind or withdraw from the GABA‐binding pocket when exposed to 500 and 380 nm illumination respectively (Figure 3C, panel a). This inhibitory effect of the agonist‐based molecule was observed previously for the nAChR (Tochitsky et al., 2012) and could be caused by a disruption of the concerted reorganization of the agonist binding site during activation and consequent conformational changes required for the opening of ion channels (Miller and Smart, 2010).

Another compound, maleimide‐azobenzene‐4‐hydroxybenzylamine (MAB)‐0, that contains a neutral analogue of muscimol and has no carbon spacer, was even more effective as a light‐sensitive inhibitor of GABA receptors. After treatment with MAB‐0, cultured hippocampal neurons expressing an α1(T125C) subunit were effectively modulated by light (Lin et al., 2014).

A series of PTLs were further developed for the efficient light‐mediated control of all α subunits (α1–α6) of GABAA receptors. For each isoform, the best PTL/mutant pair was selected based on two criteria: (i) GABA‐elicited currents are robustly photo‐controlled (preferably >50% photo‐antagonism at EC50) and (ii) receptor function is unaffected by cysteine mutation and PTL conjugation. Moreover, mutated GABA subunits were incorporated in living animals by generating a knockin mouse in which the ‘photoswitch‐ready’ version of a GABAA receptor subunit genetically replaces its wild‐type counterpart, ensuring normal receptor expression (Lin et al., 2015). This elegant approach allowed the mapping of the subcellular distribution of different α subunits in neurons and the characterization of the differential distribution pattern of GABAA receptors in the brain of living animals (Figure 3D).

In general, the team of Kramer (Lin et al., 2014; 2015) proposed a ‘toolkit’ for efficient optogenetic control of GABAA receptors. Similar to that proposed previously for the nACh and glutamate receptors (Volgraf et al., 2006; Tochitsky et al., 2012), it consists of (i) a photoswitchable‐tethered ligand composed of a cysteine‐reactive maleimide group for receptor conjugation, an azobenzene core for photoswitching and a GABA‐site ligand for competitive antagonism, such as GABA or its guanidinium analogues and (ii) the α subunits of the GABA receptor with a genetically engineered cysteine near the GABA‐binding site. This optogenetic pharmacology toolkit allows an accessible investigation of endogenous GABAA receptor function with high spatial, temporal and biochemical precision. However, antagonism of GABAergic transmission results in excitatory stimuli, which limits the applications of these switches for studying neuronal circuits and complicates the interpretation of photomanipulation experiments. Future studies should be oriented towards the development of selective optopharmacological potentiators of GABA receptor function.

Conclusion

Since the first reports almost 50 years ago, the engineering of light‐gated receptors has greatly expanded. Highly efficient photoswitches of many neuronal receptor‐channels have been reported, based on rational design, high‐resolution protein structures, comparative pharmacology and molecular biology manipulations. Several studies have demonstrated that photochromic compounds can be used for optical control of the behaviour and function of different organs. It has been shown that AzoCholine, which specifically activates neuronal nAChRs, modulates behaviour in the nematode C. elegans. The other photoswitches, AzoCarbachol, modulated in a light‐dependent fashion the beat frequency of a whole heart preparation of the mouse (Damijonaitis et al., 2015b). Photoswitches can restore electrophysiological and behavioural responses to light in mutant strains of blind mice (Polosukhina et al., 2012). Also, ATA, a freely diffusible‐specific photochromic agonist for AMPA receptors, modulated the function of amacrine and retinal ganglion cells in a light‐dependent manner, although a minor effect on bipolar cells was observed (Laprell et al., 2015). These observations suggest the powerful potential of photochromic compounds for ophthalmology.

Still, several aspects can be considerably improved. With regard to the optical properties, red‐shifted variants are not available for all photoswitches, and efficient two‐photon switching is also desirable for localized activation at the micrometre (subcellular) level. In addition, a drawback that is applicable for most PCLs and many PTLs is that their ligand action (agonist or antagonist) is exerted in the dark (trans isomer) (nAChR: Tochitsky et al., 2012; GABAA: Lin et al., 2014), and this results in the requirement to illuminate with UV light in order to maintain normal receptor activity. TCPs allow the photoswitch action to be reversed (Izquierdo‐Serra et al., 2016), although this option depends on the actual localization of the suitable reactive residues in each receptor protein.

In general, photopharmacological compounds represent efficient tools for reversible and reproducible activation or block of specific neurotransmitter‐gated receptors and ion channels in specific cells. However, subtype selectivity, which is a very desirable pharmacological property, is only found in few cases. The covalent attachment of the PTL to the target protein provides high subtype specificity compared to soluble pharmacological agents. PTLs allow precise spatiotemporal control since the photoisomerization of azobenzene is a picosecond process and binding is not limited by diffusion (Levitz et al., 2016). Successfully engineered PTLs include light‐gated glutamate receptors activated by MAG (Volgraf et al., 2006) or L‐MAG0460 (Kienzler et al., 2013), and TCP (Izquierdo‐Serra et al., 2016), and neuronal ACh receptors activated or inhibited by MAACh or MAHoCh (Damijonaitis et al., 2015a). These approaches, however, need genetic manipulation of the target protein. There is still an urgent and important need to develop highly specific soluble pharmacological agents. A combination of the expanding knowledge of crystal structure, pharmacological analysis and chemical synthesis will provide the basis for further advancements in the precision of photochromic compounds.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in The Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

We are grateful to Fabio Riefolo for his help with figure preparation. We acknowledge financial support from the ERANET SynBio MODULIGHTOR and Human Brain Project WAVESCALES projects, from the Catalan Government (CERCA Programme and 2014SGR‐1251 grant), from the Spanish Government (CTQ2016‐80066R grant), FEDER funds and from the Ramón Areces foundation.

Bregestovski, P. , Maleeva, G. , and Gorostiza, P. (2018) Light‐induced regulation of ligand‐gated channel activity. British Journal of Pharmacology, 175: 1892–1902. doi: 10.1111/bph.14022.

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