Molecular BioSystems

Optical switches and triggers for the manipulation of ion channels and pores

Pau Gorostiza^a and Ehud Isacoff*^ab
aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
^bMaterial Science Division and Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. E-mail: ehud@berkeley.edu

Like fluorescence sensing techniques, methods to manipulate proteins with light have produced great advances in recent years. Ion channels have been one of the principal protein targets of photoswitched manipulation. In combination with fluorescence detection of cell signaling, this has enabled non-invasive, all-optical experiments on cell and tissue function, both in vitro and in vivo. Optical manipulation of channels has also provided insights into the mechanism of channel function. Optical control elements can be classified according to their molecular reversibility as non-reversible phototriggers where light breaks a chemical bond (e.g. caged ligands) and as photoswitches that reversibly photoisomerize. Synthetic photoswitches constitute nanoscale actuators that can alter channel function using three different strategies. These include (1) nanotoggles, which are tethered photoswitchable ligands that either activate channels (agonists) or inhibit them (blockers or antagonists), (2) nanokeys, which are untethered (freely diffusing) photoswitchable ligands, and (3) nanotweezers, which are photoswitchable crosslinkers. The properties of such photoswitches are discussed here, with a focus on tethered photoswitchable ligands. The recent literature on optical manipulation of ion channels is reviewed for the different channel families, with special emphasis on the understanding of ligand binding and gating processes, applications in nanobiotechnology, and with attention to future prospects in the field.

With the explosive advance of structural biology and genomics, the future challenge for biology has shifted from identifying protein players and defining their individual properties to developing new ways of determining how they operate coherently in cells. Proteins do not work alone. They function in multi-component molecular machines and are coordinated in intricate interacting pathways. To understand how these signaling systems work we must develop the means to study them in living cells. This requires molecularly focused methods for dynamic interrogation and manipulation. An attractive approach is to use light as both input and output to probe molecular machines in cells. While in recent years there has been significant development in optical detection of protein function, advances in the remote control of proteins have lagged behind.

The power of rapidly turning on and off with light selected proteins in a cell has the greatest impact on the fastest signaling proteins in biology: receptors and channels. They gate in the sub-millisecond to millisecond regime and generate brief electrical and biochemical signals that govern a host of diverse biological processes, including chemosensation, hormone secretion, muscle contraction, neuronal signaling, and glucose metabolism. Optical control could advance understanding of the molecular mechanisms underlying channel function and the role of channels in cell signaling. Moreover, the ability to stimulate selected neurons in isolated tissue and in living animals would be invaluable for investigating the role of specific cells in neural circuits and in behavior.

This review is focused on the optical manipulation of ion channels and pores for the study of the kinetics and molecular mechanisms of gating, and for the production of novel nanoscale devices that are subject to remote control for use in cells, tissues and live organisms. The emphasis is on synthetic strategies for optical control of protein function using light-controlled binding of free compounds or of covalently attached ones. We will briefly discuss several recently discovered naturally occurring photosensitive proteins. Aspects that have been extensively reviewed elsewhere are excluded, such as synthetic ionophores and channels,¹ caging and photoswitching of ions and proteins other than channels,² synthesis and photochemical properties of photolabile and photoswitchable compounds,³ technological applications of photoswitches in sensors,^4,5 building of nanodevices from proteins⁶ and remote control of neuronal activity and organism behavior.^7–20 Since the focus of the review is on ion channels, transporters and metabotropic receptors studied with optical tools like ligand uncaging^21–25 will not be discussed.

Two kinds of optical manipulations are usually distinguished, according to the reversibility of the light-induced reaction: phototriggers and photoswitches. Their particularities and examples are shown in Fig. 1a–j and will be discussed separately in the next three sections, followed by a detailed account of their application for the study of gating in the major receptor and channel families. Experimentally, the effect of light on phototriggers and photoswitchable channel is usually evaluated using common electrophysiological and fluorescence recordings. These include patch clamp and calcium imaging in cell cultures expressing heterologous channels and receptors, as well as in native tissues. Validation of the observed photoeffects is usually carried out by comparison to electrical stimulation and perfusion of channel ligands, depending on the nature of the channel to be photoactivated.

	Fig. 1 Summary of molecular strategies for the manipulation of ion channels with light, with examples drawn mostly from acetylcholine and glutamate receptors. (a) A ligand-gated ion channel opens upon binding of an agonist. Examples of agonists include acetylcholine (1), carbamoylcholine (2), glutamate (3) and (2S,4R)-4-allyl glutamate (4).26 (b) Caged compounds or phototriggers comprise a photolytic group bound to an agonist that is released under illumination. A common caging group is -carboxy-2-nitrobenzyl (5), used in caged carbamoylcholine (6)27 and caged glutamate (7).28 (c) Photolytic groups like o-nitrophenylglycine (Npg) (8) can also be introduced in proteins (caged proteins).29 Photoswitchable tethered ligands or nanotoggles (d–f) include agonists, antagonists and blockers. (d) Photoswitchable tethered agonists of the acetylcholine receptor, QBr (9)30 and of the glutamate receptor, MAG (10).31 (e) Tethered channel blocker MAQ (11) used on the Shaker voltage-gated K+ channel.32 (f) Principle of operation of photoswitchable tethered antagonists. (g) Photoswitchable crosslinkers or nanotweezers with conjugation chemistries directed to amino groups (12)33 and cysteine residues (13).34 (h) Free photoswitchable agonists of acetylcholine receptors, bisQ (14)30 and glutamate receptors, GluAzo (15).35 (i) Free photoswitchable antagonist 2BQ (16).36 (j) Free photoswitchable blocker N-p-phenylazophenyl carbamoylcholine (EW-1) (17).37

A set of tools that has been extensively used to control the activity of ion channels with light involves the use of chemicals that are capable of absorbing light and undergoing an irreversible reaction, usually photolysis. These photolabile compounds, or phototriggers, are bound to the molecule of interest and employed as protecting groups that prevent specific interactions until they are photoremoved. Phototriggers can be attached to small active compounds thus sequestering them from their environment prior to release (e.g. caged ions and neurotransmitters, Fig. 1b), or to proteins at critical residues such that protein activity is enabled or disabled upon release (Fig. 1c). Proteins whose activity is enabled with light are sometimes termed caged proteins. Although this conceptual definition may be useful, it is often not significant from a chemical point of view, since an identical photolabile group can be conjugated by different means to small ligands, oligonucleotides, short peptides and full-length proteins.

The most widely employed phototriggers for ion channel studies are caged ligands that, upon photolysis with flash lamps or lasers, release channel agonists and allosteric modulators. For these applications, the desired properties include: (1) chemical stability in physiological solution and biological inertness of the caged compound (against the channel under study but also against other receptors possibly present in native preparations), (2) fast release rate, typically with half-lives below 100 µs in order to study the channel gating events that occur in the sub-ms time range, (3) high release efficacy (i.e. quantum yield or number of ligand molecules released per absorbed photon), to achieve ligand concentrations spanning the entire physiological range with minimal irradiation-induced damage of cells and tissue, and (4) biological inertness of other photoproducts besides the uncaged ligand. In addition, phototriggers with efficient multiphoton absorption are convenient for some applications, because they allow photolysis in small focal volumes using deeply penetrating long-wavelength light.

The synthetic routes, photochemical properties and photolytic mechanisms of caging groups will not be discussed as they have been recently reviewed in detail.^3,38,39 Since a great variety of compounds has been described, many of which are commercially available, mention will be made only of recent derivatives with enhanced performance relevant to studies of specific channels and receptors (e.g. ultrafast release; high release efficiency considering both quantum yield and extinction coefficient; multiphoton absorption, etc). While a universal cage (Polytechnus) may never be found,⁴⁰ several photolabile groups have been described that meet or exceed many of the requirements listed above. In practice, compromises have to be made regarding certain properties and different caged compounds can be chosen depending on the specific application desired. The 2-nitrobenzyl type cages shown in Fig. 1b⁴¹ were the first to be developed⁴² and are probably the most widely used nowadays, including commercially-available compounds like CNB- and DMNB-caged glutamate or CNB-caged GABA (see, e.g., the online catalogs of Calbiochem or Molecular Probes). More recently, caged compounds based on 7-nitroindoline like NI- and MNI-glutamate,⁴¹ coumarin-4-ylmethyl⁴³ and p-hydroxyphenacyl⁴⁴ have gained popularity despite their limited availability. An example of receptor phototriggering is shown in Fig. 2a, where activation of nicotinic acetylcholine receptors is achieved by uncaging the agonist carbamoylcholine.²⁷

	Fig. 2 (a) Patch clamp current recording of acetylcholine receptors in a BC3H1 muscle cell after laser photolysis of caged carbamoylcholine at t = 0, recorded with the cell-flow technique at –60 mV. The solid line is the observed whole-cell current, and the dotted line is the calculated corrected amplitude based on the rate of receptor desensitization.27 (b) Activation of photoswitchable ligands of the acetylcholine receptor: free photoswitchable agonist bisQ (left) and tethered agonist QBr (right). Upper plots indicate the voltage steps and UV light flashes applied to electric eel electroplaques during 3 episodes taken at intervals of 0.5 s, and lower plots show the corresponding current relaxations measured by patch clamp. For bisQ (left), at the start of the trial the solution contained 60 nM trans-bisQ and the remaining bisQ was in the inactive cis form. 15 ms after the start of the episode, the voltage was jumped from +51 to –150 mV and the agonist-induced currents increased exponentially, with superimposing time courses for the first two episodes. Halfway through the second episode, the light flash occurred, increasing the trans-bisQ concentration to 243 nM, which produces more than half-maximal activation of the agonist-induced conductance. Following this jump of trans-bisQ concentration, the conductance increased exponentially to a much larger value. The third voltage-jump relaxation occurred in the higher trans-bisQ concentration, and the rate constant and final conductance were equal to the light flash relaxation. For the experiment with the tethered agonist (right), QBr was conjugated to receptors and unbound QBr was washed away. The preparation was then exposed to UV light to convert the tethered QBr molecules to the cis photostationary state.45 Fig. 2b reproduced from The Journal of General Physiology, 1980, 75, 207–232. Copyright 1980 The Rockefeller University Press.

Although fast flow techniques⁴⁶ allow relatively rapid (sub-millisecond) ligand concentration jumps in cell cultures and excised patches with a theoretical limit of 20 µs,⁴⁷ in practice the time resolution is lower due to the sensitivity of patch seals to shear flow and the difficulty of rapid fluid exchange in large chambers containing cell cultures and native tissue.^46,48 Furthermore, perfusion of the interior of cells poses greater limitations regarding speed as well as dialysis of essential cytoplasmic components. Optical activation of receptors using rapid (up to µs) ligand uncaging thus provides a convenient and versatile alternative to flow cells, allowing high temporal and spatial resolution studies of the kinetics of receptor gating, including early events of agonist binding,⁴⁹ opening and closing rate constants, ligand dissociation constants and open probability upon ligand binding,⁵⁰ and determination of gating limiting steps and subunit cooperativity.⁵¹

Spatiotemporally designated patterns of ligand release together with assessment of activation using imaging or electrophysiology has allowed the localization of receptors and the determination of their functional role.^52,53 This has proven extremely useful in the case of complex intracellular signaling events which are difficult to perturb selectively and non-invasively, e.g. in the dissection of specific endocytic pathways.⁵⁴ More importantly, ligand uncaging has made the remote control of neuronal activity possible, using endogenous⁵⁵ and exogenous receptors.⁵⁶

The logic of using a photoisomerizable chromophore to switch the functional state of a protein is taken from nature. The classical example is the chromophore retinal in two classes of opsin protein to which retinal binds. The best understood of these are two examples of type A 7-helix transmembrane (TM) receptors: rhodopsin, a G-protein coupled receptor (GPCR) from rod photoreceptors that mediates visual transduction, and bacteriorhodopsin, a bacterial proton pump that is involved in solar energy conversion. Retinal, in its 11-cis isomer form, binds to the opsin protein in a deep binding pocket that lies in the span of the membrane, at the junction of a number of the TM helices. This location is homologous to the ligand-binding site of type A GPCR neurotransmitter and chemosensory receptors. Upon absorption of a visible photon this pre-bound ligand isomerizes from its inert 11-cis form to the all-trans conformation, which activates the opsin protein by inducing it to undergo a conformational change that is thought to be analogous to what happens when free ligand binds to the neurotransmitter and chemosensory receptors. The opsin proteins, including the recently discovered Channelrhodopsin-2 (ChR2), a cation channel,^57,58 and Halorhodopsin (Halo or NpHR), a Cl^– pump,^59,60 can be expressed heterologously in various cells, and produce fully functional proteins even without the need for exogenous supply of retinal in some cases.⁸ This renders the expressing cells sensitive to light, and thus confers the ability to influence the physiology of those cells with optical stimulation.^10,19 There has been a particular interest in using these naturally light-activated proteins to manipulate activity in neurons for circuit mapping^7,16,61 and for studies of the neural basis of behaviour.^15,17,19

The success of opsins to remotely control neuronal firing has sparked the search for other photoactivated proteins in order to exploit their natural functions. The last example is a photoactivated adenylyl cyclase (PAC).⁶² The photosensitivity of PAC is endowed by two flavin adenine dinucleotides and it has allowed the optical control of intracellular levels of cAMP.

Can the relationship between retinal and opsin be applied to other classes of protein in order to expand the repertoire of functions that can be controlled by light? The problem is that the retinal binding pocket is so complex that it probably cannot be transplanted onto another protein without transplanting the entire 7-TM helical protein. Thus, so far the only way of expanding the use of retinal has been to co-express an opsin along with both a compatible heterotrimeric G-protein and a target of that G-protein.⁶³ By its nature, this approach is limited to a small number of targets and to applications where several proteins can be heterologously co-expressed. Moreover, it is hard to prevent the opsin from coupling to other off-target proteins. Thus the challenge for engineering light sensitivity onto proteins is to find a way to take retinal, or another photoisomerizable molecule, and couple it directly onto the protein of interest, and in a manner that the protein will feel when the chromophore isomerizes.

The approach has been to covalently attach the photoisomerizable moiety site specifically to the target protein at either a native amino acid or at an amino acid that has been introduced by mutagenesis. The amino acid of choice can be a cysteine, providing the attachment selectivity of thiol chemistry, or it can be an artificial amino acid, or an amino acid sequence. The photoisomerizable moiety itself, or another component that hangs off its end, must exert a differential effect on the protein upon absorption of light. This can be achieved by capitalizing on the large structural difference between the isomers, which can change molecule length or the angle between bonds. Isomerization can exert an effect in three ways that are depicted in Fig. 1:

(1)Optical nanotoggles change the location in space of the free end of the tethered molecule. In the case where the free end bears a ligand, the geometry can be such that the ligand can only bind in one of the isomeric states. The ligand can be a blocker or an activator and the binding site can be an active site in a functional domain or an allosteric site in a regulatory domain (Fig. 1d–f). Alternatively, isomerization can involve a change in the electric properties of the photoisomerizable molecule, thereby introducing a localized partial charge that points in a particular direction and thus locally influences the protein.

(2)Optical nanotweezers are crosslinkers, which attach between pairs of residues in the protein. Photoisomerization changes the length and/or angle of the linker between the attachment sites and exerts force on the protein, thereby changing its structure or its ability to interact with partners (Fig. 1g). The optical nanotweezers can be used to directly force a protein to undergo its normal functional rearrangements, but in response to light rather than to its normal physiological signal, or, alternatively, can induce structural changes in peptides to switch between native forms that are competent to undergo signaling interactions, and denatured or disassembled conformations that are not.^33,34,64,65

(3)Optical nanokeys are free photoswitchable ligands whose affinity, rather than the local concentration, depends on light. In this case the ligand is placed very close to the photoisomerizable moiety so that the conformation of one of the isomers interferes with binding and/or activation (agonism), while the other one permits them (Fig. 1h–j).

All of the applications rely on the bistability and reversibility of the photoisomerization, and the substantially greater stability of one of the isomers, so that the system can be off in the dark, that inter-conversion between the structures can be repeated many times, and that each isomer is stable enough to accumulate significantly. Retinal absorbs light at one wavelength, photoisomerizes in one direction, and then is brought back to its original state either by thermal relaxation or by a catalytic process. By going to other photoisomerizable molecules it is possible to select ones which preferentially absorb one wavelength of light in one isomer and a second wavelength in the other isomer, and which thus make it possible to use light to actively drive the protein function both on and off. Another advantage of using the approaches of Fig. 1 is their flexibility and modularity: once the protein has been engineered with a synthetic photoswitch, tuning the properties of the switch (relaxation time, absorption spectra) or the ligand (affinity, agonism/antagonism, efficacy) does not require readjusting the protein binding pocket, as it would with natural chromophores.

While optical nanotoggles have been used to drive the gating of several ion channels and receptors (see sections below), the application of optical nanotweezers to channels has been limited to simple cases like gramicidin.³³ One possible reason is that although atomic structures of ion channels have been obtained in recent years, implementation of optical nanotweezers requires knowledge of the intrinsic stiffness of the protein domains to which the crosslinkers attach as well as of the detailed structural rearrangements that occur during gating, such that they can be effectively driven by changes in bend angle and length of the photoisomerizable molecule. Optical nanotweezers hold promise as probes of molecular rearrangement, much as scanning cysteine accessibility mutagenesis (SCAM) can be used to map solvent accessibility of individual residues in different functional states,^66,67 and voltage clamp fluorimetry (VCF) makes it possible to identify domains that move during specific gating steps.⁶⁸ The use of optical nanotweezers will benefit from molecular dynamics modeling of channels incorporating crosslinkers in aqueous environment.

The synthetic photoswitch that is most extensively used for channel applications is azobenzene.³⁰ The dark, thermally relaxed trans isomer of azobenzene adopts an extended conformation that is 7 Å longer than the cis or bent isomer. Irradiation with near-ultraviolet (UV) light (360-380 nm) induces a change from trans to cis configuration, and visible light (>460 nm) switches the azobenzene back to the trans form. Visible light (>460 nm) is absorbed better by the trans isomer and so switches the azobenzene back to the trans form. Photoisomerization cycles can be repeated many times without detectable bleaching. The dipole moment of the two isomers differ by 3 D.⁶⁹ Light changes the absorption spectrum of azobenzene such that in an ensemble of molecules, a photostationary state is established at each wavelength, corresponding to a fraction of cis and trans isomers. Typically, a population of azobenzene molecules is maximally 85% cis at the optimal UV wavelength, and the fraction is reduced to a minimum of 15% cis at the optimal visible wavelength, with virtually full conversion to the trans form only after thermal relaxation. The photoisomerization lifetime of azobenzene is in the picosecond range⁷⁰ with a quantum yield of 0.2–0.4,⁷¹ much faster than photolysis of the best caged compounds (µs), affording an advantage for rapid kinetic studies in cases where a functional readout is possible at such high speed.

The first application of the nanotoggle approach to photoswitch an ion channel was carried out in the nicotinic acetylcholine receptor (nAChR) without any structural data and even before knowledge of the protein sequence was obtained (Fig. 2b).³⁰ This achievement was based on the finding that native cysteine(s) in the vicinity of the acetylcholine binding site could be reduced and used to conjugate tethered, non-photoswitchable agonists and antagonists, thus producing constitutively active and inactive receptors.^72–74 Conjugating to the nAChR a tethered agonist of similar length, but bearing an azobenzene in the tether (QBr), allowed the agonist to bind the receptor and activate it in the trans configuration, while in the cis configuration the tether was too short for the agonist to bind (Fig. 2b). Thus the first synthetically light-sensitive receptor was obtained, although the technical limitations of the time hindered its generalization to other proteins.

More recently, a rationally designed photoswitchable glutamate receptor was described, based on the known atomic structure of the ligand binding region and the possibility of systematically testing several attachment sites in the protein using site-directed mutagenesis and heterologous protein expression.³¹ The method is outlined in Fig. 3 in the form of a flow diagram, and can be generalized to other allosteric proteins of known structure. The first step in the development of a photoswitchable receptor is the examination of the structure of the ligand binding site, to evaluate whether the bound ligand remains accessible to the solvent, or, alternatively, if the conformational changes in the binding pocket completely bury the ligand. The reason for considering this is that one prefers a case where a portion of the ligand remains accessible. From the accessible portion the tether can be extended without interfering with the rearrangements of the binding pocket that drive gating.

	Fig. 3 Flow chart describing a generalized, rationally designed approach to receptor photoswitching using tethered ligands (nanotoggles). The method was first applied to a glutamate receptor31 based on the known agonist (2S,4R)-4-allyl glutamate (4).26 A tether model compound (18) was designed, leading to the full photoswitchable tethered agonist MAG (10). In order to find the optimal attachment site for MAG, receptor mutants were generated bearing a single cysteine at different positions near the glutamate binding site (8 cysteine sites shown simultaneously in red in inset a). When conjugated to the optimal site (residue 439, above), the glutamate end of cis-MAG (shown in green) docks perfectly in the binding of the receptor (inset b).

Ideally, the portion of the ligand that is accessible to solvent would include portions known to have minimal importance to the docking interaction, so that they can serve as tethering points without sacrificing too much in terms of affinity. Based on these considerations, a tethered ligand model compound is designed and synthesized to test whether it can activate (for an agonist) or block without activating (for an antagonist). In some cases, such tether models can already be found in pharmacological compound libraries, for example affinity labels⁷⁵ and biotinylated or fluorescent ligands whose activity has already been characterized. The objective in this initial step is to find or synthesize a tethered agonist model compound that displays similar properties to those of the bare ligand when assayed in the receptor (e.g. agonist affinity and efficacy). In cases where all ligands lose their activity upon derivatization (e.g. if the ligand binding site is buried deep in the protein structure and cannot tolerate an extension), it may be possible to try open up a tether exit tunnel from the ligand binding site to bulk water by mutating residues that are in the way, although the chance of success here is probably low. Based on the best tether model compound, a full photoswitchable tethered agonist is then synthesized, including a reactive group for protein conjugation (usually reactive to cysteine) at the other end of the photoisomerizable moiety. In order to find the optimum site in the protein for photoswitch attachment, a set of receptor mutants is then generated, replacing single, solvent-exposed residues by cysteine, over a perimeter band around the ligand binding pocket where the linear distance is within range of the estimation of photoswitch length. Spacers can be added to lengthen the tether between the ligand and attachment ends. After conjugating each cysteine mutant to the photoswitch, light activation is functionally assayed and scored in a screening process, and the best responding mutant is finally selected.

In the case of the photoswitchable glutamate receptor,³¹ the tether model compound was based on a known pharmacological agonist of iGluR6, (2S,4R)-4-allyl glutamate²⁶ shown in Fig. 3, which indicated a site for extending a tether off the glutamate without lowering activity. After synthesizing the full photoswitch (termed MAG for Maleimide–Azobenzene–Glutamate), a screen of 11 cysteine mutants yielded several positions resulting in light-activation after MAG conjugation,⁷⁶ with L439C displaying the largest responses.³¹ The local effective concentrations of trans and cis ligand measured in this receptor were 0.5 mM and 12.5 mM respectively, representing a 25-fold change in effective concentration and confirming the expected photoswitching mechanism.⁷⁷

The photochemical properties of azobenzene switches have been well studied and can be adjusted with substitutions near or at the benzene rings, for example to alter thermal relaxation rates and absorption spectra.⁷⁸ Intermediate wavelengths between the maximum and minimum fractional occupancy of the cis state can be used to control in an analog manner the balance between cis and trans, and thus the percent activation of the channel.⁷⁷

Besides azobenzene, other switches like spiropyran/merocyanine⁷⁹ and hemithioindigo⁸⁰ have advantages of their own and have been applied recently. Rather than simply controlling linker length or angle, these other switches can affect protein function by reversibly altering polarity. Spiropyrans/merocyanines undergo ring opening/closing transitions upon illumination, which result in length changes of less than 3 Å but substantial changes in dipolar moment, 15 D.⁸¹ In the dark and under visible illumination, the less-polar spiropyran form is favoured. Irradiation with near-UV light causes opening of the ring to the merocyanine form, which has a higher polarity. Large changes in polarity also result from cis–trans photoisomerization of hemithioindigo compounds.⁸⁰

Several strategies have been devised to exploit light-reversible changes to activate and deactivate ion channels. These are represented in Fig. 1d–j, together with examples of compounds reported in the literature. Most of these are azobenzene-based photoswitches for nAChR and iGluR, the most extensively engineered receptors. Nanotoggles can be in the form of agonists, antagonists or blockers (Fig. 1d–f), where the ligand is attached near the binding sites in the channel with a photoisomerizable tether, such that the reduced length of one isomer, or a bend in the tether, causes a reduction of the volume sampled by the ligand, or enables the ligand to turn into the binding pocket, and thus increases the effective local concentration of the ligand in the binding pocket.⁷⁷

Nanokeys are shown in Fig. 1h–j where receptor ligands (an agonist, an antagonist and a blocker, respectively) are conjugated to a photoswitch, such that the conformational change of the switch is coupled to a reversible masking of the ligand moiety, and thus to a change in the affinity for the receptor.^30,35

In all cases, rapid jumps in the concentration of one isomer over the other are induced by illumination, making it possible to activate and deactivate the receptor with light. Further characterization of photoswitch performance includes recording the action spectra and kinetics, conjugation efficacy, extent of optical activation vs. ligand agonism⁷⁷ and action–reaction interplay between protein dynamics and photoisomerization of switch.⁸²

Another important aspect to consider with photoswitchable tethered ligands and crosslinkers is the strategy for their conjugation to the channel. Although cysteine-selective chemistry is usually the choice, unnatural amino acid technology makes it possible to introduce groups that provide for orthogonal chemical attachment or, alternatively, to directly introduce as an artificial side chain either a photoisomerizable group such as azobenzene⁸³ or a photocleavable group.²⁹ This approach can be done in an entirely genetically encoded system in bacteria⁸⁴ and has been recently applied to mammalian cells.⁸⁵

Since compounds like QBr (for nAChR) and MAG (for iGluR) have one end that reacts selectively with an extracellular cysteine and another end that has high affinity for the ligand binding site, the question arises whether ligand binding will affect conjugation to the protein. In non-photoswitchable quaternary ammonium compounds that tether to K⁺ channels, it was found that the conjugation kinetics become faster with increasing ligand affinity, suggesting that the effective concentration of the cysteine-reactive end in the vicinity of the target cysteine is enhanced when the ligand end of the molecule docks in the binding site,⁸⁶ a phenomenon known as affinity labeling. MAG photoswitch conjugation to iGluR6 demonstrates affinity labeling.⁷⁷ This can be seen in two ways. First, MAG conjugation can be interfered with by competitive binding of free glutamate in the binding pocket, which prevents the glutamate end of MAG from docking and bringing the maleimide end into proximity to the introduced cysteine. Second, photoisomerization into the non-activating conformation carries the reactive group away from the cysteine and slows conjugation. In a remarkable and powerful application of the state-dependence of affinity labeling (preferred conjugation for the activating isomer) it is possible to pattern conjugation over an otherwise uniform set of cells by patterning illumination into regions of 380 nm (favoring the cis isomer) and 500 nm (favoring the trans isomer).⁷⁷

In the oxidizing external environment of cells, cysteines often occur in disulfide-bonded pairs. Where they exist as solo residues they are usually at locations that are not important for function, where the protein can tolerate their mutation and thus, likely, their oxidation or derivatization by thiol reactive agents. As a result, external attachment of cysteine-reactive probes (including fluorophores) is usually well tolerated by cells. Consistent with this, even at high concentrations where conjugation would not be limited only to affinity labeling, MAG is not toxic for cultured neurons or zebrafish.⁸⁷ Moreover, neurons and fish that do not express the cysteine-substituted version of iGluR6 do not become sensitive to light.⁸⁷ This is despite the fact that the native iGluRs and mGluRs contain exposed single cysteines. MAG likely attaches to the native externally exposed cysteines on the GluRs and other proteins, but does unproductive light-driven calisthenics, because native cysteines are not located within the 1–1.5 nm distance of a glutamate binding site. Thus high selectivity of optical gating emerges from the strict requirement of a precise geometric relation between the attachment site and the ligand binding site, and this overcomes two features that are far from unique: the abundance of cysteines and the fact that multiple proteins are activated by the same ligand.

While the photoswitch approach has proven very advantageous to target extracellular channel domains (e.g. by perfusion of free photoswitchable ligands or photoswitchable tether conjugation by means of cysteine-reactive groups), access to intracellular domains demands higher selectivity and has not been reported. Bio-orthogonal conjugation strategies like biarsenical compounds to tetracysteine motifs⁸⁸ or derivatization of ketones, azides or alkynes introduced as unnatural amino acids^89–91 and even introduction of photoswitchable amino acids⁸³ may solve the problem for conjugation to intracellular domains.

Free photoswitchable ligands or nanokeys can be readily perfused into native receptors and tissues like caged compounds but they offer the added benefit of reversibility. Light-dependent affinity results in dose–response or dose–inhibition curves that are shifted by illumination, hopefully displaying a large separation between them which corresponds to the usable concentration range of the photoswitchable compound. However, this range is often limited due to small affinity differences between the isomers, and to the constraints in the ratio of cis : trans isomers under UV and visible illumination discussed in section IV.1. The first free photoisomerizable ligand (EW-1, Fig. 1j) was originally designed as an inhibitor of acetylcholinesterase⁹² and was later found to act as a photoswitchable blocker.³⁷ The design was soon extended to a photoisomerizable agonist (bisQ, Fig. 1h and Fig. 2b)³⁰ and antagonist (2BQ, Fig. 1i).³⁶ Although bisQ was reported simultaneously with the tethered nAChR agonist QBr, progress with free compounds was faster probably because they did not require mutation of the receptor, which was not available at the time. More recently, a fully structure-based design of free and tethered photoswitchable iGluR agonists has been reported.^31,35

As in the case of caged compounds, free photoswitchable ligands are perfused into cell cultures or tissue and thus need to be exhaustively checked against antagonist and blocker activity at high concentrations and voltages, both in the channel of interest and in other receptors that may be simultaneously present in its native context.

Photoswitching and phototriggering ion channels has provided great insights into their kinetics and gating mechanisms. In the following sections, we review some of the major studies in this area according to the main receptor and channel families, including studies carried out in vitro and in native tissue.

The nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction has been widely studied, its gating is reasonably well understood and its optical activation has been demonstrated using most of the strategies depicted in Fig. 1. Many years before the synthesis of caged compounds and even cloning of the receptor, photoactivation of nAChRs was achieved in electric eel electroplaques using a free photoswitchable agonist based on azobenzene, bisQ (Fig. 1h and Fig. 2b).³⁰ Under visible light, bisQ is predominantly in the trans conformation and acts as a full agonist. Upon isomerization to cis-bisQ under UV light, agonism is dramatically reduced. Photoisomerization and subsequent induction of nAChR currents could be achieved reversibly with millisecond light pulses. The same study took advantage of native cysteines in the vicinity of the ACh binding site in order to conjugate a tethered photoswitchable agonist, QBr (Fig. 1d and Fig. 2b).³⁰

After perfusion of the electroplaques with cis-bisQ, photoisomerization to the active trans form produced rapid agonist concentration jumps which allowed following of the time course of nAChR activation with millisecond resolution and comparison to activation with voltage-jumps.⁹³ These results demonstrated the existence of a rate-limiting process in the activation of nAChR after ligand binding. A detailed investigation of the kinetics of channel closing upon photoisomerization of bisQ to the cis form was also carried out, and was consistent with the channel closing in 100 µs after a single bound ligand is isomerized.^94,95 Gating of AChRs with reversible agonists (Fig. 1h) and tethered photoswitchable QBr (Fig. 1d) displays many similarities in channel conductance, voltage sensitivity, time course of the relaxations and in the behavior of non-competitive channel blockers, but differ in the rate of channel opening (depending only on intramolecular events in QBr-tethered AChR) and in the effect of competitive antagonists (QBr being less sensitive due to a higher effective local concentration of the agonist).⁴⁵ QBr-tethered AChRs display a similar photoswitching behavior to free bisQ, and concentration-jump and voltage-jump relaxations have also nearly equal rates. Each channel's activation is determined by the configuration of a single tethered QBr molecule. The comparison lead to the conclusion that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists.⁴⁵

Tethered and free photoswitchable agonists were further exploited to study nAChR kinetics and dose–response curves in cultured rat myoballs.^96–98 cis-BisQ, the inactive isomer, was purified^99,100 and applied to voltage-clamped whole cells or excised patches in order to record respectively macroscopic or single-channel current relaxations after light-induced concentration jumps. The open and close time constants were 7.7 ms and 0.8 ms respectively and were consistent with single-channel data.⁹⁶ Dose–response curves were also obtained in this preparation, together with evidence of a voltage-dependent block by trans-bisQ at high concentrations.⁹⁷ These studies were extended to AChR tethered agonists QBr and bromoacetylcholine (BrACh) which relaxed with time constants of 5 ms and 10 ms respectively.⁹⁸ More importantly, manipulating the ratio of cis : trans QBr isomers with light it was possible to investigate the functional stoichiometry of tethered QBr, leading to the conclusion that the open state of each receptor channel is controlled by the isomeric state of a single tethered QBr molecule.⁹⁸ As pointed out by the voltage-dependent block by trans-bisQ mentioned above, the behavior of synthetic photoswitches often turns out to be more complex than designed. For example, in vesicle preparations it was found that the purified inactive cis-bisQ isomer can actually desensitize the receptor, and that the trans form becomes an inhibitor of receptor function at progressively lower concentrations as the transmembrane voltage is decreased to more negative values.¹⁰¹ Other studies showed that bisQ behaves as a competitive antagonist in muscarinic receptors.¹⁰²

Free photoisomerizable nAChR ligands were further explored with the development of the competitive antagonist 2BQ (Fig. 1i).^36,103 2BQ shifts the agonist dose–response curves toward high concentrations, displaying apparent dissociation constants of 0.3 and 1 µM for the cis and trans isomers, respectively. At concentrations above 4 µM, 2BQ becomes a channel blocker but this behaviour is independent of the isomerization state. A free photoisomerizable channel blocker named EW-1 (Fig. 1j) was also synthesized⁹² and used to test the mechanism of open-channel blockade.³⁷ Local anaesthetics and drugs containing charged amino groups like EW-1 were thought to block ion flow through channels by binding to sites within the channel like a plug in a drain. Rapid photoisomerization of trans-EW-1 to the cis form, which is a much stronger blocker, was used to show that blockade of the open channel occurs even without previous binding of the blocker, thus providing a direct demonstration of the open-channel blockade mechanism.³⁷ These photoisomerizable ligands of nAChR and their application to understanding ion channels were reviewed ten years after the first reports.^104,105

The development of caged compounds during the 1980s led to the synthesis of a photolabile AChR agonist, carbamoylcholine (Fig. 1b and Fig. 2a).^27,106 Laser photolysis of the compound activates nAChRs without producing desensitization and only a weak interaction with the receptor prior to release.¹⁰⁶ Carbamoylcholine photolysis was used to study the activation kinetics of nAChR in muscle cells with ms time resolution.¹⁰⁷ The results could be later reproduced in muscle and neuronal AChRs expressed in oocytes, using a combination of fast flow and photolysis techniques with a resolution of 2 ms and 100 µs respectively.¹⁰⁸ Carbamoylcholine was also the first compound to be released by two-photon excitation in a biological system, allowing determination of the distribution of AChRs in muscle cells with high spatial resolution.¹⁰⁹

Another optical manipulation technique that has been used to study structure–function relationships in nAChRs is photochemical proteolysis (Fig. 1c). When the unnatural amino acid (2-nitrophenyl)glycine (Npg) is introduced in nAChR using site-specific incorporation techniques,⁹¹ irradiation of the protein results in Npg photolysis and cleavage of the peptide backbone.²⁹ The approach was first validated by photocleaving the well-characterized N-terminal inactivating peptide of the Shaker B K⁺ channel (see section below). Npg was then introduced in two sites of the nAChR: (1) a transmembrane residue known to be important for acetylcholine sensitivity, and (2) the N-terminal extracellular disulfide loop that is characteristic of the cysteine-loop superfamily but whose exact role was unclear. In both cases, electrophysiology revealed large changes in the agonist-induced currents upon Npg photolysis, resulting from the destruction of the agonist binding site and/or disruption of the gating pathway. Toxin binding assays showed structural changes in the protein only after photocleavage of the cysteine loop. These experiments provided evidence for an essential functional role of the highly conserved cysteine loop.²⁹

The incorporation into nAChRs of unnatural amino acids with caged tyrosine and cysteine side chains has also been explored using similar techniques.^110,111 Upon photolysis, the side chains are exposed and can undergo phosphorylation, disulfide bond formation or further chemical modification.

The strategies reported to optically manipulate glutamate receptor-channels (AMPA, NMDA and kainate receptors) include caged ligands, free photoswitchable ligands and tethered photoswitchable ligands. As already described, the tethered agonist approach (Fig. 1d) was combined with structure-based design to produce a light-activated glutamate receptor.^31,77 The switch rate of a population of channels in a cell is proportional to illumination intensity.⁸⁷ At an intensity of 5–8 mW mm^–2, in the middle of the working range of confocal microscopes, 1–2 ms pulses of light can activate enough iGluR6 channels to reliably depolarize and fire neurons.⁸⁷

Neither the presence of the cysteine attachment site, nor conjugation of the MAG photoswitch to the receptor alters the receptor's affinity for glutamate,⁷⁷ indicating that the photoswitch provides a true orthogonal means of receptor activation and should be useful for kinetic studies. One of the advantages of the tethered approach in heterologously expressed glutamate receptors is that it should make it possible to study the role of different subunits in heteromultimeric receptors where only a fraction of the subunits have an introduced cysteine and therefore conjugate to MAG and become subject to optical activation. In addition, the photoswitchable ligand tethering approach should also be applicable to iGluR5, NMDAR and the prokaryotic glutamate receptor homolog GluR0, each of which displays an exit tunnel in the crystal structures of their ligand bound states similar to that of iGluR6.³¹ On the other hand, attempts to obtain a photoswitchable antagonist of iGluRs, although simple in principle, have been hampered by limited antagonism of the tether models and poor solubility of the photoswitchable compounds.¹¹²

As in the case of the tethered AChR agonist QBr (Fig. 1d), the technology was extended to a free photoswitchable agonist.³⁵ The design in this case was based on the compound (2S,4R)-4-cinnamyl glutamate²⁶ which featured a cyclic ring close to the glutamate moiety that was replaced by the photoisomerizable azobenzene group. Differential activation of the compound at UV and visible wavelengths was observed in iGluR5 and iGluR6, with maximal affinity differences in the former (EC₅₀(vis) = 9 µM, EC₅₀(UV) 70 µM). Importantly, at concentrations corresponding to the maximal differential activation, the compound allowed photocontrol of depolarization and firing in untransfected cultured neurons.³⁵

Since the first reports on caged glutamate compounds,^28,113 over 20 forms have been described, as recently reviewed.¹¹⁴ Photolysis releases the ligand in tens of µs and is suitable for kinetic studies of glutamate receptors in the µs to ms time region.^28,115–117 A caged kainate compound that is inactive prior to uncaging made it possible to measure in rat hippocampal neurons the opening of endogenous kainate and AMPA receptors with a resolution on the µs timescale, yielding a rate constant of 1700 s^–1.¹¹⁸ A caged NMDA correspondingly opened NMDA receptors in neurons with a rate constant of 100 s^–1.¹¹⁹ In order to study the behavior of specific receptor subtypes, the receptor can be expressed heterologously in a cell line, such as HEK293. In this way, the kinetic constants of the AMPA receptor iGluR2Q_flip and the kainate receptor iGluR6 were measured after photolysis of caged glutamate.^50,120 The opening and closing rate constants were, respectively, 8000 s^–1 and 2300 s^–1 for iGluR2Q_flip, and 11000 s^–1 and 420 s^–1 for iGluR6. Kinetic modeling suggested that both channels open with a probability of 0.96 after binding of two glutamate molecules, which yields a shortest rise time for channel opening of 17 µs and 120 µs for iGluR2Q_flip and iGluR6 respectively. The corresponding intrinsic dissociation constants of glutamate are 1.27 mM and 450 µM respectively. These results indicate that at a glutamate concentration of 100 µM the integrated neuronal signal will be dominated by the slower iGluR6 response.⁵⁰ Other AMPA receptors were subsequently studied using the same method, and produced opening and closing rates of respectively 29000 s^–1 and 2100 s^–1 (iGluR1Q_flip) and 68000 s^–1 and 3300 s^–1 (iGluR4), values faster than previous measurements using fast solution exchange techniques.^121,122

Although the focus in the development of caged compounds is usually on optimizing their photolytic properties, it is important to verify their possible inhibitory effects on the target receptor as well as on other receptors present in native tissue.¹²³ It would also be interesting to extend these optical studies of gating kinetics to mutants with altered ligand binding and gating properties, and to well-defined (heterologously expressed) heteromultimeric receptors that mimic the subunit composition of native receptors in neurons. In this direction, a report has been published this year on the activation kinetics of NMDARs using glutamate uncaging.¹²⁴ Mutations in the ligand binding domain (LBD) of the NR2A subunit affect differentially agonist affinity and efficacy, suggesting a link between channel gating and the late steps of agonist-induced binding domain closure. Similar studies should be possible on the glycine subunit of NMDARs with a proper choice of inert glycine precursors.¹²³

An interesting application of caged glutamate has been to identify the molecular events involved in agonist binding to a glutamate receptor. Time-resolved Fourier-transform infrared (FTIR) spectroscopy has enabled the characterization of the kinetics of formation and decay of the intermediates and products involved in the photolysis of caged glutamate, with resolution on the µs time scale.¹²⁵ The method has been subsequently applied to investigate the interactions of glutamate, AMPA and kainate bound to isolated LBDs of iGluR2,¹²⁶ and to follow the time course of cage-released glutamate binding to the LBD.⁴⁹ The latter study showed that the -carboxylate of glutamate docks first into the LBD and induces changes in the protein backbone, and that interactions with the -carboxylate are established in a second step leading to locking of the agonist in the LBD.

Since glutamate is the major excitatory neurotransmitter in the mammalian brain, numerous studies have employed caged glutamate to reveal the presence of glutamate receptors in native tissue preparations,¹²⁷ and to remotely excite neurons in culture and in slices in order to investigate their connectivity and synaptic plasticity.¹²⁸ These studies take advantage of neurotransmitter uncaging in high-resolution spatiotemporal patterns together with activity imaging (e.g. using calcium- or voltage-sensitive dyes) in laser-scanning microscopes.^55,129 To this end, compounds that allow efficient glutamate uncaging with two-photon excitation have been developed, making it possible to study functional glutamate receptors with a spatial resolution corresponding to individual synapses.^130–133 These tools are leading to important breakthroughs in the understanding of excitatory input integration (REF) and long-term potentiation at the level of single dendritic spines.^134,135,136

Ionotropic -amino butyric acid (GABA) receptors (GABA_A) are inhibitory and belong to the cysteine-loop superfamily, together with nAChR, glycine and 5-HT₃ receptors. Several caged GABA compounds have been described,^137–139 but in some cases the uncaged form acts as an antagonist¹¹⁵ or blocks inhibitory postsynaptic currents in slices.¹⁴⁰ A combination of cell-flow and photolysis experiments with 30 µs temporal resolution has revealed two wildtype forms of the GABA_A receptor having different desensitization kinetics.¹⁴¹ The ligand dissociation constant and receptor opening and closing rates were determined for the two forms. An analogous approach was used to characterize the kinetics of a mutant GABA_A receptor involved in epilepsy¹⁴² as well as the mechanism of inhibition of GABA_A receptors by picrotoxin.¹⁴³

A very recent addition to the toolbox for photomodulating GABA_A receptors is a fluorescent neurosteroid that strongly potentiates receptor responses upon illumination with visible light.¹⁴⁴ Although the photopotentiation phenomenon is not yet fully understood at the molecular level, it was readily applied as an inhibitor of neuronal firing and anesthetic.

Several caged ligands of the inhibitory glycine receptor have been synthesized with progressively improved photolysis rates, stability in aqueous solution at neutral pH and biological inertness.^{31,115,145–148} Using the best behaved precursors, glycine uncaging in the 10 µs time domain together with electrophysiological recordings at 100 µs resolution provided a complete characterization of the rate and equilibrium constants of individual receptor reaction steps, including ligand binding/unbinding, activation and desensitization.¹⁴⁹ Although the inhibitory effects of several caged glycine compounds on the NMDA receptor have been evaluated,¹²³ activation of the glycine binding site of the NR1 subunit of NMDA receptors has not been studied directly.

Although most efforts in the optical manipulation of ion channels have been devoted to glutamate and acetylcholine receptors, currently there exist compounds to activate with light virtually every receptor family.

Caged ATP was first reported in 1978⁴² and there are currently about 10 different forms. Since ATP binds a great variety of proteins, caged ATP has been extensively used in mechanistic studies from ion pumps to kinases and molecular motors. However, its application to ion channels has been reported in few cases, despite the excellent kinetic properties of certain derivatives.¹⁵⁰

There are several ATP-gated ion channels, which are classified in different families. ATP-sensitive K⁺ channels (K_ATP channels) open in response to a decrease in the intracellular ATP/ADP ratio, thus coupling cell metabolism to electrical activity. Using caged ATP and pharmacological blockers, it was found that the resting membrane potential of vascular muscle cells is regulated by ATP.¹⁵¹ In another study, a K_ATP current was identified and characterized in rat striatal cholinergic interneurones.¹⁵²

P2X receptors are extracellular ATP-gated ion channels of the purinergic receptor family. Rapid ATP uncaging was used to study the fast activation and desensitization kinetics and the single channel conductance of native and fluorescently tagged P2X₃ receptors.¹⁵³ The physiological role of P2X receptors has been studied in a wide variety of tissues, including the cochlea. Real-time imaging of Na⁺ influx into cochlear hair cells combined with ATP photorelease and electrophysiological recordings made it possible to localize Na⁺ entry to the apical surface of the cell, and to follow the time course of Na⁺ diffusion through the cytoplasm.⁵² These all-optical experiments suggested a mechanism by which ATP could directly regulate the hearing sensitivity, without involving G-protein coupled P2Y receptors.

In another application, P2X₂ receptors have been over-expressed in neurons and photoactivated by means of caged ATP in order to gain remote control of neuronal activity.⁵⁶ Caged ATP is also often used to rapidly control kinase activity and has been applied to study channel phosphorylation (see section on voltage-gated channels).

Photolabile serotonin compounds were developed to study the kinetics of the excitatory 5-HT₃ receptor, the only ligand-gated channel of the serotonin receptor family.¹⁵⁴ The synthesis of these compounds was based on caged phenylephrine.^155,156 Although serotonin photorelease is relatively fast (16 µs), the low quantum yield (0.03) has only allowed partial activation of the receptors, hampering the determination of the time constants of channel opening and closing.

The recent identification of transient receptor potential (TRP) ion channels and their involvement in transducing a variety of stimuli (from light to noxious stimuli like heat, pH and capsaicin, the primary pungent compound in chili pepper) has generated enormous interest and sparked the development of molecular tools to understand the complex molecular mechanisms of these channels. Several caged capsaicin compounds have been synthesized^56,157–159 and used to photoactivate TRPV receptors. In particular, capsaicin uncaging on neurons expressing heterologous TRPV channels was used to photocontrol neuronal firing with high selectivity and at rates up to 40 Hz.⁵⁶ Novel caged capsaicin analogues can be photolyzed by two-photon excitation and allow high spatiotemporal control of ligand release and receptor activation in combination with calcium imaging.⁵³ Biological studies of TRP channels with light so far have concerned channel localization in dissociated cells and in tissue, as well as their modulation by protein kinase C phosphorylation using caged ATP.¹⁶⁰

Although cyclic nucleotide-gated (CNG) channels open in the presence of a ligand (cyclic adenosine monophosphate, cAMP, or cyclic guanosine monophosphate, cGMP), they are structurally more related to voltage-gated channels than to ligand-gated receptors. A caged cAMP compound was first synthesized 30 years ago and assayed on cAMP-dependent kinases and phosphodiesterases.¹⁶¹ Since then, a great variety of caged cAMP and cGMP compounds have been developed,^162–167 with optimized properties like nanosecond photolysis rates¹⁶⁸ and efficient two-photon absorption.^169,170 These advances have been recently reviewed.^171,172

The molecular gating mechanism of native CNG channels in retinal rods was studied in detail by rapid photolysis of caged cGMP combined with voltage steps.¹⁷³ The results made it possible to model channel activation with three sequential cGMP binding steps, the third of which limits activation kinetics at physiological cGMP concentrations. The voltage dependence of channel activation is due to the closing rate, which is faster under hyperpolarization. More recently, the activation of olfactory CNG channels was investigated with caged cAMP and caged cGMP, leading to the conclusion that these channels open in a highly cooperative way, and that the second of three binding steps switches from a very low to a very high open probability.¹⁷⁴ Using a fluorescent cGMP analogue it was possible to determine simultaneously nucleotide binding and channel activation, thus confirming that four ligands bind per channel, that binding is cooperative and that the second binding step is rate-limiting.⁵¹ The same group revisited the rod CNG channel using efficient cGMP uncaging and single channel recordings in CNGA1 homotetramers, and found that, in contrast to earlier work, gating is not rate-limited by nucleotide binding but by voltage-dependent conformational changes of the channel.¹⁷⁵ The fractional Ca²⁺ current in CNG channels was studied by calcium imaging and current influx measurements after cGMP uncaging¹⁷⁶ in order to characterize the voltage-independence of the photocurrent kinetics in retinal rods and cones.¹⁷⁷ Caged cAMP also enabled a study of the inactivation of olfactory CNG channels by Ca²⁺/calmodulin and its dependence on channel subunit composition,¹⁷⁸ as well as determination of the role of CNG channel modulation by Ca²⁺ on the complex processes of adaptation to odorants in intact olfactory cells¹⁷⁹ and to light in intact mammalian photoreceptors.¹⁸⁰

The second messengers cAMP and cGMP also control cAMP- and cGMP- dependent kinases and thus further affect channel gating by phosphorylation. However, the extent and kinetics of this modulation is only beginning to be explored, as in the study of the inhibition of K⁺ conductance by uncaging cAMP in rat hippocampal slices.¹⁸¹ Caged cyclic nucleotides have also been employed to investigate the signaling pathways involved in sperm chemotaxis, which include binding of a chemo-attractant to a guanylyl cyclase receptor protein on the surface of the sperm and intracellular synthesis of cGMP and cAMP.^182,183 This results in Ca²⁺ influx into the sperm, but the mediation of CNG channels is unclear because responses are more delayed compared to CNG channels in photoreceptors and olfactory cells.

The ultimate optical tool to control intracellular cAMP is the naturally-occurring photoactivated adenylyl cyclase (PAC) that is involved in flagellate photoavoidance.⁶² cAMP synthesis can be triggered with light with a time constant shorter than 20 ms as determined from the activation of co-expressed CNG channels. PAC can be expressed in oocytes, in mammalian cells and in flies, where it is capable of inducing changes in behavior with light.

Reengineering of CNG channels with photoswitchable tethered ligands has not been reported, probably due in part to the added difficulty of conjugating the ligand to an intracellular binding site. However, the feasibility of this idea is supported by the fact that cGMP compounds bearing bulky fluorescent moieties preserve their agonist properties,⁵¹ and thus constitute good model compounds for the synthesis of full cGMP tethers (Fig. 3). This approach, applied to labeling selected subunits, should be very useful to confirm previously proposed models and to give further insights into the cooperativity between subunits.

Voltage-gated channels are structurally organized as tetramers which can be encoded by a single protein with four homologous repeats (like voltage-dependent Na⁺ and Ca²⁺ channels) or by four identical independent subunits (like inward rectifier and voltage-dependent K⁺ channels). A number of compounds are known to bind to the pore and block the passage of ions. When tethered blockers are attached to a cysteine introduced at the extracellular side of the Shaker K⁺ channel they can produce chronic blocking.¹⁸⁴ The tether needs to be long enough for the blocker to reach the pore from the cysteine attachment site. In the first structure-based design of a light-gated ion channel, an azobenzene group was introduced into the tether, making it possible to change the tether length by 7 Å in a light-dependent manner.³² This provided for conditional blocking of the channel by reeling the blocker in and unblocking the pore in the cis state, and extending the tether so that the blocker can reach the pore and block in the trans state (Fig. 1e). Light-driven unblocking of the Shaker channel was shown to hyperpolarize neurons and inhibit their firing in response to synaptic activity.³² Furthermore, the normally inhibitory Shaker channel could be converted into an excitatory channel by mutational disruption of the ionic selectivity filter, which rendered it permeable to Na⁺, and made it possible to fire neurons in response to light—an ultimate feat of protein nanoengineering.¹⁸⁵

The voltage-gated potassium channels Shaker B and Kir2.1 have been modified with photolytic unnatural amino acids using an approach analogous to the AChR (see above). Photolysis of Npg causes the irreversible cleavage of the protein backbone (Fig. 1c). In Shaker B, the N-terminal domain forms a ball-and-chain structure that blocks the pore from the intracellular side, thus mediating fast inactivation of the channel. Npg was introduced between the N-terminal domain and the first transmembrane domain of Shaker B, such that the N terminus would be released upon Npg photolysis.²⁹ Indeed, it was found that the modified channels gated normally in the dark, and that fast inactivation could be irreversibly eliminated by photo irradiation, without affecting other gating properties.

The inward rectifier Kir2.1 channel was modified by replacing a native tyrosine residue involved in channel phosphorylation and trafficking, with a tyrosine protected with a photolabile group (Fig. 1c).⁵⁴ In the presence of active tyrosine kinases, exposing the tyrosine residue by photolysis of the protecting group triggered clathrin-mediated endocytosis without altering the conductance and gating properties of the channel. Although direct tyrosine phosphorylation could not be demonstrated, these experiments allowed a time-resolved investigation of complex intracellular signaling events and opened the possibility of spatially-resolved studies using selective photoirradiation.

Several studies have addressed the modulation of ion channels and other proteins by phosphorylation, using caged ATP. Voltage-gated K⁺ channels and their subunits are strongly regulated by phosphorylation, which usually changes the extent of channel activation. Caged ATP was used to follow channel phosphorylation at the single channel level and to characterize the changes in open probability and in the kinetics of activation and inactivation.^186,187 In the case of L-type Ca²⁺ channels, rapid increases in the intracellular concentration of Mg²⁺ and ATP enhance calcium currents as would be expected from channel phosphorylation. However, the effect cannot be prevented by blocking phosphorylation during uncaging, revealing a direct modulation of L-type Ca²⁺ channels by MgATP.^188,189

The modulation of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels by cAMP was investigated using photolysis of caged cAMP.^190,191 The studies recorded in the same cell the cAMP-induced shift in the voltage-dependence of open probability. They revealed the fast activation kinetics of HCN channels by intracellular cAMP at negative membrane potentials, indicating that cAMP acts directly on the channel and does not involve phosphorylation steps. It should be possible to extend these experiments using caged cGMP to study the modulation of HCN channels by cGMP.

As has been described so far, the optical manipulation of ion channels has provided insight into the functional mechanism as well as the means for photomanipulating cells and behavior. It also offers promise for other applications in bioengineering using bacterial channels. Prokaryotic channels have the advantage of being relatively simple and more robust (especially when they come from thermophilic bacteria), and of being easy to produce in large amounts. Gramicidin A, -hemolysin and mechanosensitive channels like MscL have been extensively characterized, their gating mechanisms are well understood and several strategies have been described to gate them with light.

Gramicidin A is a short peptide with antibiotic properties. It assemblies into the lipid bilayer of cell membranes where it forms a -helix that is not long enough to span the membrane, but after head-to-head dimerization by their N-termini it gives rise to a membrane-spanning, cation-selective pore. The strategies devised to achieve light-activation of gramicidin include tethering a blocking (ammonium) group at the C-termini of gramicidin monomers using a photoisomerizable linker, such that upon dimerization, ion passage through the transmembrane pore can be inhibited in the cis conformation.¹⁹² This approach is similar to that depicted in Fig. 1e, but with tethered blockers at both sides of the membrane. A second strategy involves tethering two monomers together by their N-termini with photoisomerizable azobenzene linkers or nanotweezers (Fig. 1g), so that the monomers are kept apart in the trans configuration, and dimerization and channel formation is favored by the cis configuration.^33,193 A third variation on the theme involves introducing unnatural amino acids with photoisomerizable side chains (azobenzene and hemithioindigo) at the first peptide residue, on the dimer interface^80,194 In this case, the change in the chromophore dipole moment that accompanies photoisomerization alters the conductance of the transmembrane pore as previously observed with non-photoisomerizable amino acids.¹⁹⁵

The pore-forming toxin -hemolysin has been extensively nanoengineered, mainly for sensor applications.¹⁹⁶ Light activation of the protein was first demonstrated by caging a residue crucial for pore assembly: photolysis of the caging group irreversibly produced an active -hemolysin pore.¹⁹⁷ More recently, conjugation of an azobenzene group to a pore-lining residue made possible photomodulating the ionic conductance of the channel, revealing the dynamics of photoisomerization at the single molecule level.⁸² These experiments, in turn, provided evidence for how conjugation to the protein affects photoisomerization: while the free cis azobenzene compound thermally relaxes to the trans form in the dark, no relaxation was observed after 8 h for single azobenzenes bound to -hemolysin, indicating a strongly stabilizing effect.

The mechanosensitive channel of large conductance (MscL) has also been recently endowed with a light gate by site-selective conjugation of a spiropyran photoswitch.⁷⁹ Previous work had shown that mutating or chemically modifying a hydrophobic site in the pore with charges results in spontaneously gating channel.¹⁹⁸ The design exploited the change in dipolar moment of spiropyran upon isomerization in order to achieve optical gating. Although the kinetics and reversibility were somewhat limited, the modified MscL allowed the remote control with light of the release of cargo from proteoliposomes.⁷⁹

These photoswitchable pores were primarily conceived as remote-controlled nanodevices for technological applications like substance sensing and drug release. Nevertheless, they also serve to confirm pore conduction and gating mechanisms, and in the case of -hemolysin they have provided the first single-molecule measurements of azobenzene photoisomerization together with evidence of its alteration by the bound protein.

Optical manipulation methods have enabled significant advances in the understanding of ion channels, receptors and pores in the last 30 years. This includes irreversible phototriggers and reversible photoswitches. The ability to turn the photoisomerizable switches on and off repeatedly makes it possible to attach them to target proteins and thus create a synthetic chemical–protein fusion whose functional state can be toggled back and forth numerous times in response to different wavelengths of light. Ligand-gated channels have been widely studied with phototrigger caged compounds. Flash uncaging allows for concentration jumps of a great variety of channel ligands with high speed, efficacy and spatial resolution. Many of these compounds are commercially available and they make it possible to study gating kinetics down to the µs time scale, to localize receptors with sub-cellular resolution and to functionally characterize intracellular signaling cascades.

While caged ligands are very versatile, their activity usually acts on multiple protein types (e.g. all the proteins that bind a ligand such as Ca²⁺ or glutamate) and once released, they must be removed by washout. Greater specificity can be obtained from light-activated chemicals that are tethered to specific proteins. For such a strategy to work, however, it is essential that the chemical can confer multiple rounds of activation. This is achieved by both natural and synthetic photoswitches. Natural photoswitches that are based on retinal have been known for a long time. A recent quest for light-sensitive proteins that can be exploited technologically has yielded a series of genetically-targeted optical actuators. The best-known example is the cation channel ChR2, which is now widely used in its wildtype form to excite neuronal activity in cell culture and in behaving animals. The effectiveness of naturally photoswitched proteins like ChR2, and its complement the inhibitory Cl^– pump NpHR, provide a strong motivation to engineer their conductance and pumping properties to generate larger currents, and their optical properties, in the manner of the engineering that has been performed on fluorescent proteins, which are now available to cover practically all ranges of the visible spectrum. While it is highly desirable to obtain variants of these retinal-based proteins that have an increased multiphoton absorption²⁰ or faster activation and relaxation rates, these improvements will likely involve modification of the chromophore and subsequent readjustment (mutagenesis) of the binding pocket. This may require some time to develop.

Channels with synthetic photoswitches were first reported even before caged compounds, but their rational design was only accomplished recently, thanks to advances in structural biology, genomics and chemical synthesis. Three design strategies have been devised: (1) Free photoswitchable ligands display light-dependent affinity or agonism and behave like reversibly caged compounds. (2) Tethered photoswitches are based on reversible changes in the local effective concentration of a ligand. (3) Photoswitchable crosslinkers have been scarcely used in channels so far, but they hold great promise as functional probes of conformational rearrangements. So far a nicotinic AChR, a kainate-type iGluR, a Kv₁ K⁺ channel, the bacterial MscL channel and gramicidin have been modified with photoswitches. The high degree of conservation of the pore architecture of diverse K⁺ channels and of the ligand binding domains of both iGluRs and mGluRs suggest that the strategies and even the chemical photoswitches that have already been made may be adapted to related channel and receptor types. Tethered ligand model compounds exist for a variety of other receptors, which makes them interesting candidates for incorporation of a photoisomerizable molecule into the tether to create a photoswitch.

Like caged compounds, photoswitches also allow rapid and reversible changes in ligand concentration, but so far kinetic studies have only been carried out with photoswitches in nAChRs. A unique advantage of tethered photoswitches is that only mutated proteins can be chemically modified with the switch, which makes it possible to specifically block or activate individual subunits in heteromultimeric receptors. One of the powerful features of synthetic photoswitches is that they are tethered to the protein surface, rather than buried the way that retinal is in a deep binding pocket, thus providing for much greater flexibility in dimension and chemical character. This enables a modular design of the photoswitch to provide for different attachment strategies and attachment sites, distinct linker moieties and lengths, and diverse ligands. The flexibility should make it possible to improve the properties of light activation to include multiphoton absorption¹⁹⁹ or multicolor switching (e.g. the development of a blue-shifted azobenzene⁷⁸). The flexibility should also permit for the development of photoswitched tethered antagonists, which have not yet been reported. Tethered antagonists could be applied to an opto-chemical isolation of neural circuits that would be more selective than electric isolation by NpHR. Other photoswitchable receptors that remain to be developed include UV-closed excitatory channels and UV-closed inhibitory channels. An advantage of synthetic switches like azobenzene is that the ratio of cis : trans molecules in the photostationary state (and thus the relative populations of open and closed channels) can be selected with the wavelength of light. However, this fact results in residual trans and cis populations even at the wavelengths of maximal cis and trans absorption, respectively, which may be a disadvantage for certain applications (e.g. if 100% trans is desirable under visible illumination in order to shut off all channels). In these cases, a solution is to use rapidly-relaxing chromophores⁷⁸ which provide an almost 100% trans population in a few hundred milliseconds.

These optical stimulation methods have made it possible to control neuronal activity with single bouton and single action potential accuracy, as well as driving behavior in transgenic animals. The generalization of techniques like FRAP, FLIP and photoactivation of GFP derivatives using laser scanning microscopes²⁰⁰ as well as the development of sub-millisecond, sub-micrometre systems for the generation of spatiotemporally selected patterns of light like digital diaphragms and ultrafast laser beam steering systems,⁵⁵ are paving the way to all-optical experiments directed at interrogating the physiological role and cellular cycle of individual biomolecules in single cells and in the complex context of living tissue. Last but not least, photoswitches are playing a pivotal role in nanobiotechnology and nanomedicine as externally accessible switches that allow the exploitation of naturally occurring, evolution-optimized nanomachines (from channels and pores to pumps, motors and enzymes) in order to build artificial production systems, drug delivery vehicles with remotely controlled release, and smart biosensors.

Acknowledgements

We are grateful to F. Tombola for comments on the manuscript, and to J. A. Farrera for providing us with reference 71. P. Gorostiza acknowledges support by postdoctoral fellowships from the Human Frontier Science Program (HFSP) and the Nanotechnology Program of the Generalitat de Catalunya, and by a Career Development Award of the HFSP.

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Footnote

Present address: Institució Catalana de Recerca i Estudis Avançats (ICREA) and Institut de Bioenginyeria de Catalunya (IBEC), Parc Científic de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain.