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. 2016 Feb 23;110(4):896–911. doi: 10.1016/j.bpj.2015.12.033

Figure 6.

Figure 6

State-dependence of the T1 zinc bridge. (A) Patch-clamp experiment showing five test pulses (2 + 3) of 10 mM glutamate (blue bars) in the presence of 10 μM EDTA (green bars), flanking the application (1 s) of 10 μM zinc (red) with different concentrations of glutamate (indicated by the black bar): 10 μM (orange trace), 500 μM (light blue), 10 mM (dark blue). CTZ (100 μM) was present throughout the experiment. Middle panel (zoom of the top panel) shows relaxation observed at 500 μM and 10 mM glutamate (τ = 35 ± 2 ms and τ = 32 ± 9 ms, n = 6–9 patches, respectively). The active fraction is indicated in the inset with an orange triangle. The recovery of current in 10 mM glutamate and EDTA following trapping at different concentrations showed a common fast component (τ = 3 ± 0.4 ms, n = 6 patches) corresponding to the exchange of solution and a slow component (τ =106 ± 30 ms, n = 6 and τ = 147 ± 14 ms, n = 6 patches, following trapping in 500 μM and 10 mM glutamate, respectively). WT A2 shows no modification by zinc in 500 μM and 10 mM glutamate, bottom panel. (B) Glutamate concentration-response curves in 10 μM zinc for WT GluA2 (blue circles; EC50 = 170 ± 40 μM), T1 before trapping by zinc (green diamonds; EC50 = 348 ± 80 μM), and T1 following trapping (red circles; EC50 = 6.4 ± 2.9 mM). The difference between the apparent affinities for glutamate for WT and T1 in the absence of zinc was not significant (p = 0.2). The relationship between the active (untrapped) fraction and log-concentration of glutamate followed an exponential function (yellow triangles). (C) Correction for chelation of zinc by high glutamate. Blue trace shows response of mutant T1 to 500 μM L-glutamate in the presence of 10 μM zinc (red bar), flanked by 10 mM L-glutamate test pulses in EDTA (green bars). Orange trace shows the application of 29.5 mM D-glutamate in addition to 500 μM L-glutamate and 10 μM zinc. Following the application of zinc, a larger active fraction (as estimated from the larger instantaneous current activated by 10 mM glutamate) was observed in the presence of 29.5 mM D-glutamate than in its absence (inset arrow), presumably due to zinc chelation. (D) The chelation effect at 30 mM glutamate reduced trapping by 9 ± 2%. Thus, the trapped fraction was underestimated due to a lower free zinc concentration. The 30 mM glutamate value for the active fraction in Fig. 6B, was reduced accordingly by a factor of 1.09. To see this figure in color, go online.