Photosensitive tools for neuronal control can also be rationally designed and manufactured through synthetic chemistry. The general strategy is to couple a photoisomerizable molecule (i.e. a “photoswitch”) onto an ordinary ion channel or receptor to make it sensitive to light. In theory, the photoswitch can be attached in such a way that photoisomerization exerts force on the channel, causing it to open. Alternatively, the photoisomerization could deliver or remove a ligand from a binding site on the channel or receptor, thereby regulating its activity. In practice, the photoswitchable ligand approach has worked nicely with voltage-gated K+ channels and glutamate receptors. However, in theory, the photoswitchable ligand approach could apply to virtually any ion channel or receptor, as long as there are known ligands that regulate activity.
There are several chemical photoswitches available, but the photoisomerizable small molecule azobenzene has emerged as best suited for biological applications. In darkness, azobenzene exists in a linear trans configuration, but 380 nm light promotes transition to the bent cis configuration, which is ~7Ά shorter. In darkness, the cis form relaxes slowly back to the trans form (over minutes), but this relaxation can be accelerated by exposure to 500 nm light. Azobenzene compounds are relatively easy to synthesize, have well-defined geometries, and show high photochemical stability and little phototoxicity.
Erlanger and colleagues were the first to apply the photoisomerizable ligand approach to control the activity of a receptor [44
]. They synthesized a soluble photoisomerizable molecule, Bis-Q, and a cysteine-reactive derivative QBr, both of which activate the nicotinic acetylcholine receptor (nAChR) in the trans
configuration but not in the cis
configuration. QBr covalently attaches to the nAChR, but only after reducing disulfide bonds between native cysteine residues. QBr was particularly useful for rapidly delivering and removing the ligand to minimize desensitization of the nAChR, enabling detailed study of the mechanisms of receptor activation.
The molecular biology revolution has allowed investigators to take the photoswitchable ligand approach one step further. Instead of relying on a native cysteine, a particular channel or receptor can be targeted for photoswitch attachment by genetically engineering a cysteine into the appropriate location on the protein. The first step is to identify a ligand that can be modified so that it can be conjugated to the azobenzene without losing its ability to bind and regulate channel activity. Structural information about ion channels and receptors can guide the engineering of the target protein, in particular the position of the cysteine attachment site.
This approach was first used to generate a Synthetic Photoswitchable Azobenzene-Regulated K+
channel (SPARK) [46
]. SPARK channels are generated by coupling a photoswitchable ligand, maleimide-azobenzene-quaternary ammonium (MAQ), onto a genetically engineered Shaker K+
channel. The M
aleimide is for cysteine tethering, the A
zobenzene is for photoswitching, and the Q
uaternary ammonium group blocks the pore of the Shaker channel. The channel is only blocked when MAQ is in its extended trans
form, and not in the shorter cis
form. MAQ enables control of action potential firing only in those neurons that express the cysteine-containing Shaker channel. Visible light blocks SPARK channels, allowing action potential firing. UV light retracts the pore blocker, promoting the flux of K+
through the channel, which hyperpolarizes the neuron and inhibits action potential firing. A mutation that alters the ionic selectivity of the K+
channel changes the polarity of the effects, enabling depolarization and induction of action potentials with UV light [47
Glutamate receptors are another class of ion channels where photswitchable tethered ligands have been successfully applied. In 2006, a light-gated ionotropic glutamate receptor (LiGluR) was introduced [48
]. This system is based on a glutamate derivative covalently attached to a genetically engineered kainate receptor (iGluR6) via an azobenzene tether. In its original embodiment, the tethered neurotransmitter was presented to the clamshell-like binding site in the cis
configuration (380 nm light) and retracted in the trans
configuration (500 nm light). Changing the attachment site reversed the polarity, with 500 nm turning the receptor on and 380 nm light turning it off [49
]. LiGluR and its modifications can be employed to control neural activity in vitro
and in vivo
]. Very recently, LiGluR has proven to be a valuable tool for the dissection of neural circuits that control behavior in zebrafish [60
The first-generation light-activated K+ channel (SPARK) and glutamate receptor (LiGluR) were designed specifically for light-induced neuronal inhibition and excitation. Each of these photoswitch-ready channels was derived from a particular generic channel, chosen because of prior structure-function information and favorable properties. However, there is no reason why many other K+ channels and glutamate receptors would not become photoswitchable, if a cysteine attachment site were included in the correct position on the channel. For example, we have now generated several light-regulated K+ channels, including Kv3.1 and SK2 (unpublished results). This gives neurobiologists optical tools for selectively regulating functions carried out by different K+ channels. Indeed, given sufficient motivation by chemists and neurobiologists, the photoswitchable tethered ligand approach should be applicable to many other types of voltage-and ligand-gated channels.
SPARK channels and LiGluR, like ChR2 and NpHR, are genetically-encoded tools and therefore can be targeted to particular types of neurons by selective gene expression. The neuronal specificity that comes from genetic targeting is often a big advantage, but in some cases exogenous gene expression is not practical and may not even be desirable (e.g., in humans). This has motivated the development of small molecule photoswitches that act on native channels or receptors without requiring exogenous gene expression. We have developed a family of azobenzene-containing molecules that photosensitize a wide variety of native voltage-gated K+
]. We have shown that one of these molecules, AAQ, imparts light-sensitivity on neurons in cell culture, in brain slices, and in intact retina. Light-elicited blockade of K+
channels can cause membrane depolarization, leading to action potential firing. Unlike the genetically-encoded tools that enable light to over-ride the normal activity of the neuron, AAQ enables light to alter the intrinsic excitability of the neuron. Hence the properties that are regulated by K+
channel activity, including action potential threshold, propensity for repetitive firing, and spike afterhyperpolarization, can also be regulated by light in AAQ-treated neurons.
AAQ and most analogs block K+
channels when the molecule is in the trans
form (500 nm light) and unblock in the cis
form (380 nm light). However, a related molecule named PrAQ, blocks in the cis
and unblocks in the trans
form. Hence, this molecule should allow light to regulate neuronal firing in the opposite manner to AAQ [52
]. AAQ-mediated photocontrol of neurons persists for up to 24 hours after treatment, and it was initially proposed that AAQ covalently attaches to K+
channels. However, related photoswitch molecules lack a reactive group and yet still impart light-sensitivity on native K+
channels, suggesting that they linger in or near the channels without covalent attachment. Whatever the mechanism of photosensitization, these tools share in their ability to impart light-sensitivity without involving the introduction of exogenous genes.
An azobenzene-containing photoswitch has also been developed that enables photoregulation of native glutamate receptors [53
]. This “reversibly caged” glutamate (Glu-Azo) was shown to act on kainate receptors and reversibly trigger action potential firing in dissociated hippocampal neurons. Although its reversibility might be considered an advantage over classical caged glutamate, its usefulness in brain slices and live animals remains to be demonstrated.