Designing Chemical Photoswitches
Designing Chemical Photoswitches
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- We invent photochemical tools (photoswitches) containing the light-sensitive chemical azobenzene.
- Some of our photoswitches act on genetically-enginered ion channels or receptors.
- Some act on endogenous ion channels, native to the nervous system.
- Ongoing studies are aimed at optimizing photoswitches for controlling neuronal signaling, both as scientific tools and as potential drugs.
Photocontrol of genetically-specified channels
How it works: The photoswitch compound has maleimide, for coupling to a cysteine, azobenzene, for changing length with light, and a ligand (quaternary ammonium), which blocks the pore of voltage-gated channels. The channel protein has a cys mutation at the precise location where the attached photoswitch can reach the pore. Photoswitching light shortens or lengthens the azobenzene, promoting or alleviating pore block.
Photocontrol of endogenous channels
How it works: Fortuitously, we discovered that some photoswitches pass into cells though large-pore channels. Once inside, they act on native voltage-gated ion channels, with no need for genetic modification. Photoswitching light shortens or lengthens the azobenzene, again promoting or alleviating pore blockade, but in this case from the cytoplasmic side.
Photocontrol of GABAA Receptors
Photocontrol of GABAA Receptors
- GABA is the main inhibitory neurotransmitter in the brain. The synaptic receptors for GABA are ligand-gated chloride channels called GABAA receptors.
- We have generated a comprehensive toolkit, including genetically engineered proteins and chemical photoswitches, for photocontrolling each of the GABAA isoforms in the brain.
- By generating knock-in mice, we can photocontrol endogenous GABAA receptors that native to the nervous system to better understand their functions.
Photocontrol of Acetylcholine Receptors
Photocontrol of Acetylcholine Receptors
- We have generated PTLs that enable activation or inhibition of neuronal nicotinic acetylcholine receptors (nAChRs) with light.
- To target individual types of nAChRs in individual types of neurons, we engineer a cys mutation in the beta-subunit of the receptor, and use viral vectors to express the receptor in a cell-specific fashion.
- The combination of genetic targeting and lght-sensitivity gives allows us to control receptor function with unprecedented temporal, spatial, and biochemical precision.
Photocontrol of Specific Potassium Channels
Photocontrol of Specific Potassium Channels
- Potassium channel photoswitches contain a cysteine-reactive maleimide (M), a photoisomerizable azobenzene (A), and a quaternary ammonium (Q) a blocker of the pore of K+ channels.
- We genetically engineer potassium channels to contain an extracellular cysteine attachment site, precisely positioned to allow pore block once the photoswitch, MAQ, attaches.
- By photoisomerizing the tethered photoswitch from trans to cis, the pore becomes unblocked, and K+ can flow.
- By varying light stimuli, we can explore the subcellular, cellular, or network functions of specific K+ channels.
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