Neurons have ion channels that are directly gated by voltage, ligands and temperature but not by light. Using structure-based design, we have developed a new chemical gate that confers light sensitivity to an ion channel. The gate includes a functional group for selective conjugation to an engineered K + channel, a pore blocker and a photoisomerizable azobenzene. Long-wavelength light drives the azobenzene moiety into its extended trans configuration, allowing the blocker to reach the pore. Short-wavelength light generates the shorter cis configuration, retracting the blocker and allowing conduction. Exogenous expression of these channels in rat hippocampal neurons, followed by chemical modification with the photoswitchable gate, enables different wavelengths of light to switch action potential firing on and off. These synthetic photoisomerizable azobenzene-regulated K + (SPARK) channels allow rapid, precise and reversible control over neuronal firing, with potential applications for dissecting neural circuits and controlling activity downstream from sites of neural damage or degeneration.Natural photoreceptive proteins, such as rhodopsin, have a covalently attached chromophore that directly activates the protein on exposure to light. Several strategies have been used to enable light regulation of proteins that are not intrinsically light sensitive. Light can be used indirectly to activate receptor proteins, for example, by making a ligand available from a caged precursor 1 . Light can also be used to directly photoisomerize a synthetic molecule that is covalently attached to a protein, thereby imposing conformational changes 2,3 . We have combined these ideas by synthesizing a photoisomerizable tether that attaches a specific ligand on a protein near its normal binding site. Photoswitching of the tethered ligand rapidly regulates its ability to reach its binding site, thereby switching the protein on and off without causing unnatural conformational changes. Photoswitchable tethered ligands can be agonists (for example for nicotinic acetylcholine receptors 4 ), antagonists, or other regulators of protein function. We have applied this general design principle to an ion channel, where the chosen ligand is a blocker of the channel's pore. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diffusion kinetics. Recently, the exogenous expression of genes encoding ion channels has been used to influence electrical activity in specific neurons 7-9 , but the onset and reversal of gene expression is slow. A modification of this technique, in which a receptor from one species is introduced into another that lacks a natural ligand, dramatically improves temporal control 10,11 , but this approach still relies on a diffusible ligand whose time course of removal limits reversibility. Finally, photic regulation has been conferred on neurons by introducing a rhodopsin-based signal transduction cascade 12 . This technique requires coordinated exogenous expression of three different genes and produ...

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Light work: Studies into the mechanism of AAQ, a photoswitchable blocker of voltage‐gated K+ channels, led to the discovery and development of photochromic ligands that act at the internal tetraethylammonium binding site (see picture). These molecules can be applied from the extracellular side to impart long‐lasting photosensitivity on K+ channels in living cells and thereby afford photocontrol of action potential firing in neurons.

Photochromic channel blockers provide a conceptually simple and convenient way to modulate neuronal activity with light. We have recently described a family of azobenzenes that function as tonic blockers of K(v) channels but require UV-A light to unblock and need to be actively switched by toggling between two different wavelengths. We now introduce red-shifted compounds that fully operate in the visible region of the spectrum and quickly turn themselves off in the dark. Furthermore, we have developed a version that does not block effectively in the dark-adapted state, can be switched to a blocking state with blue light, and reverts to the inactive state automatically. Photochromic blockers of this type could be useful for the photopharmacological control of neuronal activity under mild conditions.

Abstract. The acronym SePhaChARNS, for "selective pharmacological chaperoning of acetylcholine receptor number and stoichiometry," is introduced. We hypothesize that SePhaChARNS underlies classical observations that chronic exposure to nicotine causes "upregulation" of nicotinic receptors (nAChRs). If the hypothesis is proven, (1) SePhaChARNS is the molecular mechanism of the first step in neuroadaptation to chronic nicotine; and (2) nicotine addiction is partially a disease of excessive chaperoning. The chaperone is a pharmacological one, nicotine; and the chaperoned molecules are α4β2* nAChRs. SePhaChARNS may also underlie two inadvertent therapeutic effects of tobacco use: (1) the inverse correlation between tobacco use and Parkinson's disease; and (2) the suppression of seizures by nicotine in autosomal dominant nocturnal frontal lobe epilepsy. SePhaChARNS arises from the thermodynamics of pharmacological chaperoning: ligand binding, especially at subunit interfaces, stabilizes AChRs during assembly and maturation, and this stabilization is most pronounced for the highest-affinity subunit compositions, stoichiometries, and functional states of receptors. Several chemical and pharmacokinetic characteristics render exogenous nicotine a more potent pharmacological chaperone than endogenous acetylcholine. SePhaChARNS is modified by desensitized states of nAChRs, by acid trapping of nicotine in organelles, and by other aspects of proteostasis. SePhaChARNS is selective at the cellular, and possibly subcellular, levels because of variations in the detailed nAChR subunit composition, as well as in expression of auxiliary proteins such as lynx. One important implication of the SePhaChARNS hypothesis is that therapeutically relevant nicotinic receptor drugs could be discovered by studying events in intracellular compartments rather than exclusively at the surface membrane.