Figure 5: PTL conjugates to GluK1 by affinity labelling.

(a–c) PTL conjugation to GluK1 is reduced by a competitive ligand. (a,b) Current recordings of GluK1-expressing cells incubated with compound 9 (2.5 μM, 10 min at pH 8) in the absence (a) or the presence of competitive antagonist DNQX (1 mM) (b). Green, violet and black bars indicate illumination at 500 nm, 380 nm and no illumination, respectively. Glutamate responses (300 μM, red bar) were used to normalize photocurrents to GluK1 expression level. (c) Quantification of normalized photocurrent difference after incubation in compound 9 with DNQX (orange, n=8; black is control, n=5). (d–g) PTL conjugation is favoured under green illumination. (d) Cells were conjugated to PTLs under green light (left) or under violet light (right). (e) Normalized photocurrent difference produced by compound 9 and 10 (left and right panels, respectively) incubated under green and violet light (respectively 0.018±0.002, n=4 and 0.08±0.02, n=4 for compound 9, and 0.027±0.009, n=6 and 0.11±0.02, n=7 for compound 10). Significance calculated by Mann–Whitney U-test, **P<0.01. (f) Compound 9 conjugation is favoured in regions of the coverslip illuminated with green light (through a × 40/0.65 objective from below the sample, whereas a violet LED lamp was applied from above; see Methods). (g) Normalized photocurrent difference produced by compound 9 in green- and violet-illuminated regions (respectively 0.08±0.02, n=3 and 0.04±0.008, n=6). Significance calculated by Mann–Whitney U-test, *P<0.05. All bars are mean±s.e.m.