Figure 1. α‐mannosidase inhibition reduces myc‐GluK2a molecular weight and alters myc‐GluK2a‐containing receptor desensitization.

A, kifunensine inhibits α‐mannosidase I and swainsonine inhibits α‐mannosidase II, blocking hybrid and complex oligosaccharide formation, respectively. B, myc‐GluK2a was expressed in untreated control, swainsonine‐treated or kifunensine‐treated HEK293T/17 cells, detected by immunoblotting with an anti‐myc antibody, and myc‐GluK2a MW was measured from gel migration. C, representative current traces from untreated control, swainsonine‐treated and kifunensine‐treated myc‐GluK2a‐expressing HEK293T/17 cells. Grey bar indicates glutamate (10 mm) application. Amplitudes are scaled. D, quantification of glutamate‐evoked desensitization and current amplitude from untreated and treated myc‐GluK2a‐expressing HEK293T/17 cells. E, representative current traces in two‐pulse glutamate (10 mm) recovery experiments recorded from untreated and swainsonine‐treated myc‐GluK2a‐expressing HEK293T/17 cells. Intervals between glutamate exposures range from 50 ms to 2 s in the traces shown. Amplitudes of the first glutamate application are scaled. F, quantification of mean glutamate recovery for myc‐GluK2a‐expressing cells with and without swainsonine. 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 τ_rec values differ significantly (P < 0.0001). G, representative myc‐GluK2a deactivation current traces. Grey bar indicates glutamate (10 mm) application. Amplitudes are scaled. H, quantification of glutamate‐evoked deactivation from myc‐GluK2a‐containing outside‐out patches pulled from untreated and swainsonine‐treated HEK293T/17 cells. I, representative current traces in recovery experiments recorded from untreated and kifunensine‐treated myc‐GluK2a‐expressing HEK293T/17 cells, as in (E). J, quantification of mean glutamate recovery for myc‐GluK2a‐expressing cells with and without kifunensine, as in (F). Results were fitted with a single component exponential equation and the best‐fit τ_rec values differ significantly (P < 0.0001). K, representative myc‐GluK2a deactivation current traces. Grey bar indicates glutamate (10 mm) application. Amplitudes are scaled. L, quantification of glutamate‐evoked deactivation from myc‐GluK2a‐containing outside‐out patches pulled from untreated and kifunensine‐treated HEK293T/17 cells. 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]