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. 2023 Dec 1;147(5):1837–1855. doi: 10.1093/brain/awad403

Figure 2.

Figure 2

Variant effects on receptor desensitization and activation properties. (A) Representative currents evoked by sequential 10–20 s applications of Glu (1 mM, black bar) alone and in the presence of cyclothiazide (CTZ) (100 μM, grey bar) from oocytes expressing wild-type (WT) GluA3 and GluA3 carrying selected GRIA3 missense variants. The p.(Pro302Ser) variant shows no change in the size of the desensitized current relative to the non-desensitized Glu current compared to wild-type, the p.(Ala654Val) variant shows increased desensitized current, and the p.(Thr816Ile) variant show decreased desensitized current. (B) Representative currents evoked by sequential 10–20 s applications of Glu (1 mM, black bar) and kainic acid (KA) (300 µM; blue bars) in the presence of CTZ (100 μM, grey bar) from oocytes expressing wild-type GluA3 and GluA3 containing selected variants exemplifying different types of variant effects on KA/GLU response ratio. For wild-type GluA3 and the p.(Pro302Ser) variant, the KA-evoked current has an amplitude of 16% of the Glu current amplitude. In contrast, the p.(Ala654Val) variant has a relative KA current of 41%, indicating an increase in activation properties, and p.(Ala653Thr) variant has decreased relative KA response amplitude of 3.5%, indicating decreased activation properties. The holding potential was −40 mV in all shown recordings. (C) Representative currents illustrating 1-naphthyl acetyl spermine (NASPM) (1 µM, red bar) inhibition of Glu-evoked currents for wild-type GluA3 and GluA3 containing the variants p.(Arg631Ser) and p.(Ala654Pro). (D) Summary of the ratio of desensitized and non-desensitized current amplitude (IGLU/IGLU+CTZ), non-desensitized Glu and KA (IKA+CTZ/IGLU+CTZ) current amplitudes and NASPM inhibition of Glu-evoked current for homomeric GluA3 receptors containing genetic variants encoded by the GRIA3 variants evaluated in this study. Values, number of measurements and statistical parameters are given in Supplementary Table 2. Individual data-points are colour-coded according to the effect on currents or EC50 [loss-of-function (LoF) effect; red] or increase [gain-of-function (GoF) effect; green]. (E–G) Summary of phenotype and domain location of variants with overall GoF (E), LoF (F) and neutral (G) effect on homomeric GluA3 receptor function. Inverted triangle = decrease; triangle = increase; filled circle = no change; dash = not determined. Colour-coding indicates a predicted LoF (red) or GoF (green) effect of change on overall receptor function. (H) Missense tolerance ratio (MTR) of GRIA3 variants analysed with a 31 amino acid window calculated using the MTR-viewer online tool (https://biosig.lab.uq.edu.au/mtr-viewer/).67 A line graph displays the MTR distribution for GRIA3 (gene transcript NM_000828) with regions in orange indicating observed variation differs significantly from neutrality. Dashed lines on the plot denote gene-specific MTRs: green = fifth percentile; purple = 25th percentile; black = 50th percentile. Above the MTR distribution is shown the domain structure of the GluA3 subunit. Variant positions are shown as circles on the MTR line graph and coloured according to functional effect as: neutral (grey), GoF (green) and LoF (red). Orange line segments indicate regions where the observed variation differs significantly from neutrality. In all panels, variants are labelled with single-letter amino acid codes. ABD = agonist binding domain; CTD = carboxy-terminal domain; NTD = N-terminal domain; TMD = transmembrane domain.