Selective Reduction of Ca2+ Entry Through the Human NMDA Receptor: a Quantitative Study by Simultaneous Ca2+ and Na+ Imaging

Figures

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  Fig. 1. Simultaneous Ca2+ and Na+ imaging discriminates between ligand-gated ion channels with different Ca2+ permeability. A representative Ca2+ transients recorded in Hek293T cells expressing human α3β4 nAChR (red) and NMDAR (green), elicited respectively by ACh, and NMDA plus glycine. B representative Na+ transients recorded in Hek293T cells expressing human α3β4 nAChR (red) and NMDAR (green), same cells as A. C FCa/FNa for NMDAR (green, n = 14) and α3β4 nAChR (red; n = 16; *p < 0.001, one-way ANOVA). D linear relationships of the distributions of FCa and FNa values, for NMDAR (green) and α3β4 nAChR (red). Same cells as C

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  Fig. 2. Simultaneous Ca2+ and Na+ imaging discriminate between different Ca2+ permeation due to changes in [Na+]o. A, left, representative Ca2+ transients recorded in Hek293T cells expressing human NMDAR, elicited by coapplication of NMDA and glycine, in 0 mM [Ca2+]o (grey), 1 mM [Ca2+]o (blue) and 2 mM [Ca2+]o (green). A, right, representative Na+ transients recorded in Hek293T cells expressing human NMDAR, same cells as A. B FCa/FNa for NMDAR in the three conditions of [Ca2+]o (0, 1 and 2 mM, n = 8, 15 and 14, respectively; *p < 0.001). C linear relationships of the distributions of FCa and FNa values for NMDAR in the three conditions of [Ca2+]o

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  Fig. 3. Simultaneous Ca2+ and Na+ imaging is not able to detect the effect of extracellular mild acidosis on NMDAR Ca2+ permeability. A representative Ca2+ transients recorded in Hek293T cells expressing human NMDAR, elicited by coapplication of NMDA and glycine in control cells (green, pH 7.4) and cells in a 6.5 pH extracellular solution (red). B representative Na+ transients recorded in Hek293T cells expressing human NMDAR, same cells as A. C FCa/FNa for NMDAR in control cells (green; n = 14) and in cells exposed to a 6.5 pH extracellular solution (red; n = 12); no significant difference. D linear relationships of the distributions of FCa and FNa values for NMDAR in control cells (green) and in cells exposed to a 6.5 pH extracellular solution (red). Same cells as C

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  Fig. 4. EU1794-4 reduces the Ca2+ permeability of human NMDAR. A representative Ca2+ transients recorded in Hek293T cells expressing human NMDAR, elicited by coapplication of NMDA and glycine in control cells (green) and in cells treated with EU1794-4 (30 μM, red). B representative Na+ transients recorded in Hek293T cells expressing human NMDAR, same cells as A. C FCa/FNa for NMDAR in control cells (green; n = 14) and in cells treated with EU1794-4 (red; n = 81). D linear relationships of the distributions of FCa and FNa values for NMDAR in control cells (green) and in cells treated with EU1794-4 (red). Same cells as C

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  Fig. 5. EU1794-4 reduces the fractional Ca2+ current (Pf) of the human NMDAR. A representative traces of the whole-cell currents elicited by the coapplication of NMDA and glycine in control cell (green) and in cell treated with EU1794-4 (30 μM, red). B representative traces of the Ca2+ transients recorded simultaneously with the current traces shown in A. At an excitation wavelength of 380 nm, [Ca2+]i increase corresponds to a downward deflection of Fura-2 fluorescence emission. Traces in A and B are aligned and share the same temporal scale. C linear relationships between ΔF/F (data shown in panel B) and Q (time integral of the whole-cell current shown in panel A, calculated at the same times of ΔF/F values) obtained from the same cells as in A, B. The dashed line represents the mean slope obtained from calibration experiments (5.54 nC.−1; n = 9). D, box plot of Pf values in control cells (green; n = 8) and in cells treated with EU1794-4 (red; n = 8; *p = 0.025)

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  Fig. 6. EU1794-4 reduces NMDAR-mediated Ca2+ influx in human neurons derived from iPSCs, in the presence or absence of Mg2+. A representative Ca2+ transients elicited by coapplication of NMDA and glycine in human neurons before (left), during (center) and after (left) treatment with EU1794-4 (in Mg2+-free solution). B box plot of the NMDA-induced FCa values in the presence (green; n = 15) or absence of 2 mM extracellular Mg2+ (red; n = 16) in control cells (empty boxes) and in cells treated with EU1794-4 (hatched boxes; a: p = 0.011, b: p < 0.001). C box plot of the normalized NMDA-induced FCa values in the presence (green; n = 15) or in the absence (red; n = 16) of 2 mM extracellular Mg2+ in neurons treated with EU1794-4 (c: p < 0.001)