Design of an allosteric photoswitch. (a) An agonist (orange) is tethered to an LBD through an optical switch (red) via linkers (black). In one state of the switch, the ligand cannot reach the binding pocket, whereas in the other state, the ligand docks and stabilizes the activated (closed) conformation of the LBD. (b) Schematic representation of the operating mode of iGluRs. Binding of an agonist (orange) stabilizes the activated (closed) conformation of the LBD and allosterically opens the pore, allowing flow of Na⁺, Ca²⁺ and K⁺. NBD: N-terminal domain; TMD: transmembrane domain. (c) The principle of LiGluR. Reversible optical switching of a tethered agonist on the LBD opens and closes the pore.

Structures and fit of photoswitched agonist and iGluR6 LBD. (a) Chemical structure of the iGluR6 agonists (2S,4R)-4-methyl glutamate 1; (2S,4R)-4-allyl glutamate 2; and tether model 3. (b) Structure of MAG 4 in its trans state (dark and 500 nm) and cis state (380 nm). (c) View looking into the ‘mouth’ of iGluR6 LBD in complex with 1 (ref. ). Residues on clamshell ‘lips’ that were individually mutated to cysteine are highlighted in yellow. Position 439 is shown in red. The methyl group of 1 can be seen in blue at the bottom of the ‘exit channel’. (d) Docking model of MAG in the cis state attached at L439C (yellow) and bound to the activated (closed) conformation of the LBD.

Calcium imaging of iGluR6 activity. (a) Superimposed bright-field (gray) and enhanced yellow fluorescent protein (EYFP; green) images of HEK293 cells transfected with both iGluR6 and EYFP. (b) Calcium image (350/380 nm) of FURA-2-AM–loaded cells during perfusion of 300 μM glutamate. Red and blue correspond to high and low Ca²⁺ concentrations, respectively. (c) Simultaneous Ca²⁺ concentration traces from individual cells in response to indicated concentrations of the tether model 3. (d) Dose-response curves from Ca²⁺ traces as in c. Higher concentrations of model 3 activate iGluR6 to levels similar to those resulting from saturating concentrations of glutamate (1 mM). Responses were normalized with respect to saturating concentrations of glutamate. Tether model 3 has an EC₅₀ of 180 μM, compared with the higher-affinity molecule 2 on which it was based, which has an EC₅₀ of 18 μM. (e) MAG confers light sensitivity to iGluR6-L439C–expressing cells, as seen by reversible increases in intracellular Ca²⁺ concentration at 380 nm and decreases at 500 nm. MAG does not confer light sensitivity to wild-type iGluR6-expressing cells (WT). Agonism by free MAG is transient and reverses upon washout for both iGluR6-L439C and wild-type iGluR6. IGluR6-L439C retains ability to be activated by free glutamate after MAG 4 conjugation. Note that Ca²⁺ concentration was not measured during irradiation at 380 or 500 nm or during conjugation.

Total synthesis of MAG 4. Reagents and conditions: (a) Boc-Gly-OH, EDCI, HOBt, DIPEA (66%); (b) TFA, CH₂Cl₂ (98%); (c) see ref. ; (d) acrylic acid, 5% Grubbs' second-generation catalyst (92%); (e) H₂, Pd/C, MeOH (97%); (f) 6, EDCI, HOBt, DIPEA (97%); (g) Fmoc-Gly-OH, (COCl)₂, DMF; (h) piperidine, DMF (43%, over two steps); (i) 1.0 M LiOH H₂O/THF, 0 °C (80%); (j) N-methoxycarbonylmaleimide, NaHCO₃, THF/H₂O (71%) and (k) HCl-saturated EtOAc (87%). Boc, t-butoxycarbonyl; CH₂Cl₂, dichloromethane; (COCl)₂, oxalyl chloride; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; EDCI, N-ethyl-N′-(3-dimethyldiaminopropyl)-carbodiimide HCl; EtOAc, ethyl acetate; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole hydrate; MeOH, methanol; Pd/C, palladium on carbon; TFA, trifluoroacetic acid; THF, tetrahydrofuran.

Whole-cell patch-clamp current recordings from HEK293 cells expressing iGluR6-L439C after conjugation of MAG. (a) Inward currents (downward deflections, carried mainly by Na⁺ influx) in response to glutamate are preserved in LiGluR. Irradiation with short wavelengths of light (280–480 nm, in 20-nm increments) results in maximal activation at 380 nm. Irradiation with long wavelengths of light (400–600 nm, in 20-nm increments) leads to maximal deactivation at 500 nm. Alternation between 380- and 500-nm illumination evokes highly reproducible responses. (b) Patch-clamp traces comparing the effects on LiGluR of a saturating concentration of glutamate, optical switching and the titration of the tether model 3. Saturating responses elicited by 3 are slightly higher than those elicited by 380-nm irradiation, suggesting geometric constraints that prevent the LBD from fully closing on MAG.