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. 2021 Aug 9;108(9):1692–1709. doi: 10.1016/j.ajhg.2021.07.007

Figure 3.

Figure 3

Functional and biochemical characterization of homomeric mutant GluK2 KARs

(A) Representative whole-cell currents evoked by a 1–2 ms application of glutamate (10 mM, gray bar) from recombinant receptors composed of the indicated subunits expressed in HEK293-T/17 cells. The holding potential was −70 mV in all recordings. Note that the GluK2(A657T) and GluK2(T660K/R) mutant receptors exhibited two temporally distinct phases to the current decay.

(B) Quantitation of the decay tau values fitted either to one exponential function (WT and I668T) or two components weighted by proportional contributions (A657T and T660K/R).

(C) Mean peak amplitude of glutamate-evoked currents. Note the semi-log y axis.

(D) Representative whole-cell currents evoked by a 1 s application of glutamate (10 mM, gray bar) from recombinant receptors composed of the indicated subunits.

(E) Quantitation of desensitization tau values fitted to two exponential decay functions weighted by proportional contributions.

(F) Quantitation of percent desensitization for glutamate-evoked currents.

(G) Representative traces in which two glutamate applications were made with variable intervals for generation of the time course of recovery from desensitization. Glutamate was applied for 50 ms in these experiments.

(H) Quantitation of recovery from desensitization data. Tau values from the fits are given in the text.

(I) Quantitation of GluK2 KAR membrane expression in HEK293-T/17 cells. Receptor-expressing cells were exposed to biotin followed by isolation on streptavidin-conjugated beads, separation on SDS-PAGE gels, and immunoblotting with anti-GluK2/3 or anti-actin antibodies. Samples shown are input (“In”), cytosol (“Cy”), and surface (“S”). Wild-type GluK2 and GluK2(I668T) proteins are predominantly found on plasma membrane (S) relative to intracellular compartments (Cy), whereas surface localization of A657T and T660K/R proteins is reduced. Biotin was omitted in the control (“Ctrl”) samples. The graph shows quantification of the data via densitometry normalized to actin staining. Molecular weight markers are shown in kDa. Statistical significance is denoted as ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. p values are also given in the text. WT, wild-type; wtd, weighted. Error bars represent SEM.