Extended Data Fig. 4: Current-voltage (IV) and conductance-voltage (GV) plots confirm that GluA2-containing AMPARs are polyamine insensitive and kinetic properties corroborate CNIH presence in the AMPAR complex.
From: GluA2-containing AMPA receptors form a continuum of Ca2+-permeable channels
a. IV plots of different AMPAR-TARP + CNIH complexes in external 150 mM Na+ (open circles fitted by a 4th order polynomial function) solution. b. GV plots of the AMPAR-TARP complexes obtained from IV curves above (a). Open circles show average normalized response and colored shading show se.m. (A1γ2/A2 + CNIH-2: n = 9; A1/A2γ2 + CNIH-2: n = 8; A1γ2/A2 + CNIH-3: n = 6; A1/A2γ2 + CNIH-3: n = 9). c. Example membrane currents evoked by 10 mM L-Glu (250 ms duration) on GluA1/A2 AMPARs co-assembled with either TARP γ2 and CNIH2 or TARP γ2 and CNH3 (c). The grey shadow shows the SEM of the response. d. Comparison of gating properties between A1/A2γ2 (in grey) vs A1/A2γ2 + CNIH-3 (black) receptors. e. CNIH-3 slows desensitization kinetics of A1/A2γ2 receptors (A1/A2γ2: τdes = 7.5 ± 0.6 ms, n = 16; A1/A2γ2 + CNIH-3: τdes = 22.5 ± 1.7 ms, n = 9). Equilibrium current was enhanced by CNIH-3 (A1/A2γ2: Equilibrium current = 2.9 ± 0.4 %, n = 16; A1/A2γ2 + CNIH-3: Equilibrium current = 14.7 ± 1.3 %, n = 9). CNIH-3 also slowed the off-kinetics of AMPARs (A1/A2γ2: τoff = 6.8 ± 0.5 ms, n = 19; A1/A2γ2 + CNIH-3: τoff = 23.5 ± 1.5 ms, n = 14) and the deactivation kinetics (A1/A2γ2: τdeact = 1.5 ± 0.1 ms, n = 6; A1/A2γ2 + CNIH-3: τdeact = 7.0 ± 0.8 ms, n = 9). Two-sided unpaired t-tests with Welch correction. p-value *** <0.001. f. AMPAR-TARP + CNIH complexes arranged by the reversal potential observed in 108 mM external Ca2+ solution (A1/A2: ErevCa2+ = −44.8 ± 2.8 mV, n = 6; A1γ2/A2 + CNIH-3: ErevCa2+ = −33.6 ± 1.7 mV, n = 6; A1γ2/A2 + CNIH-2: ErevCa2+ = −25.8 ± 2.2 mV, n = 9; A2γ2/A3 + CNIH-3: ErevCa2+ = −29.7 ± 2.2 mV, n = 5; A1γ8/A2 + CNIH-3: ErevCa2+ = −31.2 ± 2.7 mV, n = 5; A1/A2γ8 + CNIH-3: ErevCa2+ = −18.9 ± 3.4 mV, n = 6). Orange square shows the A1/A2γ2 AMPAR to highlight changes in the Ca2+ reversal potential induced by CNIH-2 and -3 auxiliary proteins (cyan squares, A1/A2γ2 + CNIH-2: ErevCa2+ = −1.8 ± 2.5 mV, n = 8; A1/A2γ2 + CNIH-3: ErevCa2+ = 7.5 ± 1.1 mV, n = 9). Sky-blue circle denotes A1/A2γ2 + CNIH-3 with the A789F mutation which left-shifts the Ca2+ reversal potential (A1AF/A2γ2 + CNIH-3: ErevCa2+ = −25.5 ± 3.1 mV, n = 8). g. Pooled data of PCa/PNa from the AMPAR complexes shown in (f) grouped by CNIH and TARP type (A1γ2/A2 + CNIH-2: PCa/PNa = 0.15 ± 0.02, n = 9; A1/A2γ2 + CNIH-2: PCa/PNa = 0.55 ± 0.11, n = 8; A1γ2/A2 + CNIH-3: PCa/PNa = 0.10 ± 0.01, n = 6; A1/A2γ2 + CNIH-3: PCa/PNa = 0.95 ± 0.05, n = 9; A1AF/A2γ2 + CNIH-3: PCa/PNa = 0.16 ± 0.02, n = 8; A1γ8/A2 + CNIH-3: PCa/PNa = 0.12 ± 0.02, n = 5; A1/A2γ8 + CNIH-3: PCa/PNa = 0.23 ± 0.04, n = 6; A2γ2/A3 + CNIH-3: PCa/PNa = 0.12 ± 0.02, n = 5). Two-sided Kruskal-Wallis ANOVA followed by Mann-Whitney U tests with Bonferroni-Holmes correction. p-value * <0.05, ** <0.01, *** <0.001.
