Figure 6. Reversible NMDAR photopotentiation with PSAA at various pore sites.

(a) Structure of the GluN1/GluN2 TMD represented by a side (left panel) and top view (right panel). The four sites of the hydrophobic cluster, where PSAA was incorporated, are illustrated as spheres in different colors. (b) Example recording for GluN1-647PSAA/GluN2A mutants. UV illumination in the presence of the co-agonists resulted in a 3.4-fold potentiation of receptor activity. (c) Same cell as in b, with light applied in the resting state. Here, the UV-driven potentiation was 3.9-fold. (d) Same cell as in b, with light applied in the ‘mixed’ states. In this example, the current increased by 5-fold. (e) As is b, for the GluN1-654PSAA/GluN2A mutant. Here, UV light induced a 4.6-fold potentiation. (f) The fold-potentiation of activity differed between the mutants, but not between their functional states. On average, the photopotentiation in the active state is: Pot_F554 = 1.4 ± 0.1 (n = 5), Pot_W563 = 2.3 ± 0.1 (n = 3), Pot_Y647 = 4.3 ± 0.4 (n = 14), Pot_F654 = 3.9 ± 0.2 (n = 12). UV in the resting state resulted in Pot_Y647 = 3.4 ± 0.2 (n = 5) and Pot_F654 = 3.6 ± 0.5 (n = 6); and in the ‘mixed’ state in Pot_Y647 = 3.9 ± 0.3 (n = 8) and Pot_F654 = 3.7 ± 0.5 (n = 6). The dashed line indicates a potentiation of 1 (i.e. no potentiation). (g) Kinetics of photopotentiation. The mean photopotentiation exponential time constants are (in ms): τ_F554= 1640±170 (n = 4), τ_W563 = 240 ± 20 (n = 3), τ_Y647 = 1020 ± 80 (n = 14), τ_F654 = 290 ± 30 (n = 8). (h) Example trace showing that the UV-induced potentiation, once initiated, is highly stable even after UV light has been turned off (here, for 70 s). (i) Example trace demonstrating the stability of the photoresponse over numerous trans-cis illumination cycling events.

DOI: http://dx.doi.org/10.7554/eLife.25808.017

Figure 6—figure supplement 1. Multiple sequence alignment in the TMD region across multiple iGluR family subunits (NMDA, AMPA, and kainate receptor subunits).

A section of the entire sequence around the TMD is shown. The TMD is shown in blue, the helices pre-M1 and M1 to M3 are highlighted as blue boxes. The four highly conserved GluN1 positions of the TMD hydrophobic cluster that were tested in this study are highlighted in red. All subunits are from rat.

DOI: http://dx.doi.org/10.7554/eLife.25808.018

Figure 6—figure supplement 2. No or minor unspecific Amber codon suppression at TMD sites.

(a) Example traces from GluN1-Y647Amber/GluN2A receptors incubated either with (black trace) or without (green) PSAA in the cell culture medium. No unspecific suppression of the Amber codon by endogenous amino acids was detected for this mutant. UV and blue light were equivalently applied in the –PSAA and +PSAA conditions. In the presence of the PSAA, a typical reversible photopotentiation was observed (in this example, 3.8-fold current potentiation). In the absence of the PSAA, the blank recording did not show any increase of current upon UV light. (b) As in a, for the GluN1-F654Amber mutant. A minor unspecific background in the minus PSAA-condition (–PSAA) was detected. Crucially, this ‘leakage’ current did not show any light-related effects, in contrast to receptors harboring the PSAA at the F654 site (here, 3.7-fold current potentiation). (c) Pooled peak current amplitudes for both mutants (48 hr post transfection) incubated in the presence or absence of PSAA. Supplementing the medium with the PSAA resulted in average current amplitudes of 84 ± 35 pA (n = 10) for GluN1-Y647Amber/GluN2A receptors. Only a few cells gave no agonist-mediated currents (n = 3 blanks). In the absence of the PSAA, no currents were detected in all cells tested (n = 12 blanks). For the GluN1-F654 Amber mutant, the current amplitudes were 196 ± 142 pA (n = 8) when PSAA was supplemented. The amount of blank recordings was minor (n = 1 blank). Cells lacking the PSAA gave mean current amplitudes of 150.5 ± 48 pA (n = 6), six further cells did not express at all (n = 6 blanks). UV or blue light never modulated mutant receptor activity with unspecifically incorporated endogenous amino acids. In contrast, receptors showed a 3.2 ± 0.3 fold potentiation with PSAA at the Y647 position (n = 3), and a 4 ± 0.2 fold potentiation when PSAA was introduced to the F654 site (n = 4).

DOI: http://dx.doi.org/10.7554/eLife.25808.019