Figure 4.

ZnR/GPR39 triggers SNAP23 interaction with KCC2 to enhance KCC2 membrane expression and activity in primary hippocampal neurons

(A)Mouse hippocampal neuronal cultures (DIV 15) loaded with the pH-sensitive fluorescent indicator BCECF (1 μM) were treated with Zn²⁺ (100 μM, 15 s) and rates of NH₄⁺ transport (following application of 5 mM NH₄Cl) were compared to control. To study the role of IKK phosphorylation of SNAP23, neurons were pretreated with the IKK inhibitor BAY11-7085 (5 μM), and rates of Zn²⁺-dependent NH₄⁺ transport were determined. Right panel shows averaged rates of NH₄⁺ transport, calculated over initial 100 s from maximal signal. (Box and Whisker plots, line indicates median, each dot represents the rate averaged over at least 10 neurons from an independent slide, ∗∗p<0.05 Independent-Samples Kruskal-Wallis Test).

(B) BCECF fluorescent signal traces from hippocampal neurons pretreated with a cell-permeable SNAP23-binding peptide or with a scrambled (SCR) control (3 μM). Cells were treated with Zn²⁺ or without, as in (A). Right panel shows averaged rates of NH₄⁺ transport. (Box and Whisker plots, line indicates median, each dot represents the rate averaged over at least 10 neurons from an independent slide, ∗∗p<0.05 Independent-Samples Kruskal-Wallis Test).

(C) Representative confocal fluorescence microscope images (60x) following PLA between KCC2 and SNAP23, in mouse hippocampal neuronal cultures that were treated with Zn²⁺ (100 μM, 30 s) or control. Red puncta are PLA immunolabeling, DAPI fluorescence (blue) marks nuclei. (Scale bar is 50 μm). Quantification of PLA puncta is shown on right panel (dots are quantification of single images from three independent experiments, ∗∗p<0.05, t test).

(D) Hippocampal slices were treated with Zn²⁺ (200 μM, 1 min) or without it. Membrane fractions were immunoprecipitated with an antibody against SNAP23. Lysates were then separated on SDS-PAGE and subjected to immunoblotting with antibodies against KCC2, SNAP23, or Syntaxyin 1A to determine if these proteins were co-immunoprecipitated.

(E) Hippocampal tissue slices were treated with Zn²⁺ (200 μM, 1 min) or controls. Representative immunoblots of membrane-enriched fractions and total lysates that were exposed to antibodies against KCC2, SNAP23, or Syntaxin 1A. Right panel shows boxplot quantification (line at mean) of membrane expression of KCC2, SNAP23, or Syntaxin 1A following Zn²⁺ treatment, normalized to total protein in the same fractions, dashed line notes the membrane expression level of the proteins in non-treated slices. (n = 3 independent experiments, # <0.05 Independent-Samples Kruskal-Wallis test compared to non-treated).Data are presented as box and whisker (min. to max.) plots with median marked by the line, or in bar graphs as mean ± SD, all data points are presented.