The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry.azobenzene ͉ ion channel ͉ photoisomerization ͉ optical switch ͉ remote control M uch progress has been made recently in the real-time noninvasive detection of protein function (1), but the development of approaches for remote protein manipulation within the complex environment of the cell has lagged behind. A major advance has been the development of photolysable cages for soluble ligands (2). The caged ligand is allowed to slowly diffuse into tissue in its inert form, and a powerful light flash cleaves a photolabile protecting group, releasing the active ligand rapidly (within microseconds) (3,4). This provides fast on-rates that are complemented with reasonably fast off-rates, which depend on native binding affinity, diffusion, sequestration, and breakdown. However, because native ligands often act on multiple proteins, this approach has limited selectivity. Introducing foreign receptors that bind nonnative ligands, on the other hand, enables cellular stimulation without the activation of endogenous proteins (5, 6) and can even be used in a photolysable form for rapid release (7).Photoisomerizable moieties have also found use in the remote and selective control of native protein function. In this case, reversible photochemically induced changes in the shape or electronic character of functionally important amino acids have been used to control the function of proteins in response to light (8-10) or to alter the backbone structure of peptides (11), thereby controlling their interaction with other biological macromolecules (12). In an alternative strategy, photoisomerization of a tethered ligand can be used to reversibly present, and withdraw, a ligand from a binding site. To date, several ion channel photoswitches have been reported wherein a ligand is tethered to the channel via a linker containing a photoisom...

The design, synthesis, and biological evaluation of a photochromic glutamate analogue is described. The molecule functions as a reversibly caged neurotransmitter and can be used to influence neural activity with light.