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. 2017 Aug 2;595(17):5913–5930. doi: 10.1113/JP274790

Figure 2. Glycan chains attached between the ATD and LBD are critical modulators of KAR desensitization.

Figure 2

A, western blot showing myc‐GluK2a MW after PNGase F treatment. Myc‐GluK2a‐expressing cells were incubated in buffer (untreated) or buffer containing PNGase F (live PNGF) for 2 h prior to lysing. Protein from untreated cell lysates was denatured, digested with PNGase F (den. PNGF) and run as a positive control for PNGase F‐mediated glycan removal. B, representative traces of currents evoked from untreated (control) or live PNGase F‐incubated (PNGF) HEK293T/17 cells expressing myc‐GluK2a. Grey bar indicates glutamate (10 mm) application. C, quantification of whole‐cell glutamate‐evoked desensitization and amplitude from untreated and PNGase F‐incubated cells expressing myc‐GluK2a. D, cartoon depiction of the mutated glycosylation sites in a GluK2ΔNG5,6,7 subunit. E, western blot showing GluK2ΔNG5,6,7 MW after PNGase F treatment, as in (A). F, representative current traces from untreated control, swainsonine‐treated and kifunensine‐treated GluK2ΔNG5,6,7‐expressing HEK293T/17 cells. Grey bar indicates glutamate (10 mm) application. Amplitudes are scaled. G, quantification of glutamate‐evoked desensitization and amplitude from untreated and treated GluK2ΔNG5,6,7‐expressing HEK293T/17 cells. H, representative current traces in two‐pulse glutamate (10 mm) recovery experiments recorded from untreated and kifunesine‐treated GluK2ΔNG5,6,7‐expressing HEK293T/17 cells. Intervals between glutamate exposures range from 50 ms to 2 s in the traces shown. Amplitudes from the first glutamate application are scaled. I, quantification of mean glutamate recovery for GluK2ΔNG5,6,7‐expressing cells with and without kifunensine treatment. Amplitude of the second glutamate application in a two‐pulse experiment is reported as a normalized percentage of the first glutamate application. Results were fitted with a single component exponential equation and the best‐fit τrec values differ significantly (P < 0.0001). con, control; sw, swainsonine treatment; kif, kifunensine treatment. Error bars indicate the SEM. Statistical significance is indicated: *** P < 0.001. [Color figure can be viewed at wileyonlinelibrary.com]