Stephanie Szobota

Remote Control of Neuronal Activity with a Light-Gated Glutamate Receptor

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.

Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor.

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.

A light-gated, potassium-selective glutamate receptor for the optical inhibition of neuronal firing

Genetically targeted light-activated ion channels and pumps make it possible to determine the role of specific neurons in neuronal circuits, information processing and behavior. We developed a K+-selective ionotropic glutamate receptor that reversibly inhibits neuronal activity in response to light in dissociated neurons and brain slice and also reversibly suppresses behavior in zebrafish. The receptor is a chimera of the pore region of a K+-selective bacterial glutamate receptor and the ligand-binding domain of a light-gated mammalian kainate receptor. This hyperpolarizing light-gated channel, HyLighter, is turned on by a brief light pulse at one wavelength and turned off by a pulse at a second wavelength. The control is obtained at moderate intensity. After optical activation, the photocurrent and optical silencing of activity persists in the dark for extended periods. The low light requirement and bi-stability of HyLighter represent advantages for the dissection of neural circuitry.

Optical Control of Neuronal Activity

Advances in optics, genetics, and chemistry have enabled the investigation of brain function at all levels, from intracellular signals to single synapses, whole cells, circuits, and behavior. Until recent years, these research tools have been utilized in an observational capacity: imaging neural activity with fluorescent reporters, for example, or correlating aberrant neural activity with loss-of-function and gain-of-function pharmacological or genetic manipulations. However, optics, genetics, and chemistry have now combined to yield a new strategy: using light to drive and halt neuronal activity with molecular specificity and millisecond precision. Photostimulation of neurons is noninvasive, has unmatched spatial and temporal resolution, and can be targeted to specific classes of neurons. The optical methods developed to date encompass a broad array of strategies, including photorelease of caged neurotransmitters, engineered light-gated receptors and channels, and naturally light-sensitive ion channels and pumps. In this review, we describe photostimulation methods, their applications, and opportunities for further advancement.

Optical control of ligand-gated ion channels.

In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation of this approach are covalently attached photochromic tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding spatial and temporal resolution. Here, we describe all steps towards the successful development and application of PTL-gated ion channels in cell lines and primary cells. The basis for these experiments forms a combination of molecular modeling, genetic engineering, cell culture, and electrophysiology. The light-gated glutamate receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves as a model.

Nanosculpting reversed wavelength sensitivity into a photoswitchable iGluR

Go ). Then, these fusion proteins were co-expressed with -opioid receptor (DOR), and channel 2/ 1 and 1 subunits in baby hamster kidney cells and Xenopus oocytes. Ba2+ currents through the expressed channels, their I-V relationships and G protein-mediated inhibition of the channels via DOR stimulation by the agonist enkephalin in 1B-FP and FP-Go were similar to those obtained from wild-type 1B and Go . The ratio of acceptor/donor fluorescent intensity was measured after donor and acceptor crosstalk was subtracted. In a conventional epifluorescence microscope, the ratio of acceptor ( 1B-YPet)/donor (CyPet-Go ) intensity was gradually increased by enkephalin during 8 min. Next, using a total internal reflection fluorescence microscope, characteristics of one-step photobleaching were identified as single protein molecules on the cell membrane. Enkephalin inhibited the lateral mobilities of both 1B-EGFP and EGFP-Go to 50% by 3 min, and the inhibited mobilities almost fully recovered by 5–10 min. In the single-molecule FRET configuration, enkephalin enhanced the ratio of acceptor (TagRFP-Go )/donor ( 1B-EGFP) intensity during 3 min, and this enhancement was decreased thereafter. These results indicate that receptor stimulation-induced enhancement of the interaction between G protein and N-type Ca2+ channel is reproducible at the single-molecule level.