Figure 2.

ZnR/GPR39-dependent upregulation of KCC2 activity is not mediated through its phosphorylation

(A) Scheme of residues that are putative phosphorylation sites on KCC2.

(B) BCECF fluorescent signal traces from HEK293 cells co-transfected with GPR39 and WT KCC2 or KCC2 mutant S940A, treated with Zn²⁺ (200 μM, 2 min) and compared to controls. Cells were treated with NH₄Cl and rates of acidification following NH₄⁺ transport were determined. Bar graph shows averaged rates of NH₄⁺ transport, calculated over initial 100 s from maximal signal.(∗∗∗p<0.0001 Mann-Whitney test).

(C) BCECF fluorescent signal traces from HEK293 cells co-transfected with WT KCC2 and GPR39, pretreated with the PKC inhibitor staurosporine and then with Zn²⁺, compared to controls. Right panel shows averaged rates of acidification following NH₄⁺ transport in control cells or cells treated with the PKC inhibitors staurosporine (1 nM) or GO6983 (10 μM). (∗∗∗p<0.0001 KCC2 with and without Zn²⁺, ∗∗p = 0.0022 staurosporine with and without Zn²⁺, ∗∗∗p = 0.0005 GO6983 with and without Zn²⁺, Mann-Whitney test).

(D and E) Averaged rates of acidification following NH₄⁺ transport monitored in HEK293 cells co-transfected with GPR39 and the indicated KCC2 mutants. Cells were treated with Zn²⁺ or without it. (all comparisons are between control and Zn²⁺-treated mutants, panel D using t test: p = 0.0046 KCC2, p = 0.0212 T1007E, p = 0.0088 T906E; for panel E, t test was used: p<0.0001 KCC2, p = 0.0002 S988A, p = 0.0002 S986/988A, p = 0.0003 S986/988D, p<0.0001 T584A, p = 0.0002 S868A, p = 0.0274 SPVSS; and p = 0.0041 S986A using Mann-Whitney test). All bar graphs values are averages ± SD, with all data points presented.