To selectively photo-potentiate GluN2B-NMDARs, we envisioned two strategies: one using soluble photoswitchable polyamines (SPPs), which can act on endogenous receptors but can have off-target effects (WP1); and one using tethered photoswitchable polyamines (TPPs), which have almost no off-target effect but require genetic engineering of the receptor (WP2).
Work Package 1: Selectively enhancing GluN2B-NMDARs with SPPs
In collaboration with the Baigl Lab (Ecole Normale Supérieure, Chemistry Department), we had previously designed a photoswitchable spermine, called AzoSp, that combined (i) a spermine moiety that acts as the potentiating ligand; and (ii) an azobenzene moiety that acts as the photoswitch. Azobenzenes can indeed switch from an extended, trans configuration to a bent, cis configuration following illumination with UV light, and back to trans following illumination with visible light. Like its parent compound spermine, AzoSp selectively potentiated GluN2B-containing NMDARs at low concentration, but also induced a very strong inhibition of all NMDAR subtypes by binding to the ion channel pore. The concentrations at which AzoSp induced potentiation and inhibition were not different enough to selectively potentiate GluN2B-NMDARs while avoiding inhibition of the other NMDAR subtypes. We therefore decided to stop working with SPPs and rather focus on Work Package 2.
Work Package 2: Selectively photo-enhancing GluN2B-NMDARs TPPs.
We designed a cysteine-reactive, photoswitchable spermine, called MASp, by attaching to AzoSp a cysteine-reactive maleimide. To covalently attach MASp to the GluN2B subunit, we substituted residues of the spermine putative binding site into cysteines and labeled the cysteine-mutated receptors with MASp. Out of the 23 substitution positions tested on the GluN2B subunit, 5 showed a >30 % photomodulation of the NMDAR current. One position in particular yielded up to 5-fold UV-induced potentiation after MASp labeling. Photomodulation at this position was reversible, reproducible, and allosteric, meaning that it occurred only when the receptor was activated by the agonists. This latter aspect is important, since it allows respecting the temporal pattern of receptor activation in vivo. We chose this mutant as the photo-switchable receptor to introduce in native preparations (WP3). Importantly also, labeling of GluN1/GluN2A receptors, the other major class of NMDARs in the brain, with MASp did not yield any photomodulation. We have therefore reached our goal, which was to selectively and reversibly photo-enhance GluN2B-NMDARs.
This work was presented as a poster at the Young Researcher In Life Sciences conference (Paris, May 2017).
Work Package 3: towards photo-enhancement of GluN2B-NMDAR activity in native tissues
We are currently in the process of characterizing the photomodulation of our photoswitchable receptors in HEK cells and neurons to test for the volatility of the approach and off-target effects. Our next step will be to introduce the mutated receptor in hippocampal slices and investigate the effect of potentiating GluN2B-NMDARs on several physiological processes like synaptic transmission, synaptic plasticity and excitotoxicity.
