FIGURE 8.

Regulation by acidic pH of the Zn²⁺-dependent signaling is eliminated in HEK293 cells expressing the D313A mutant. A and B, AKT and ERK1/2 phosphorylation in HEK293 cells expressing either WT GPR39 or D313A mutant were monitored after application of Zn²⁺ (100 μm, 2 min) in Ringer's solution at pH 7.4 or 6.5. Immunoblots using antibodies against phospho- or total-AKT (A) and ERK1/2 (B) are shown (upper panels). Phosphorylated protein levels were normalized to the total protein and are presented as percentage of the phosphorylation induced by Zn²⁺ at pH 7.4 (lower panels; n = 4; *, p < 0.05 compared with WT control; #, p < 0.05 compared with D313A control). C and D, ZnR-dependent up-regulation of NHE activity was monitored in HEK293 cells expressing WT GPR39 (C) or D313A (D) using the NH₄Cl prepulse paradigm. Intracellular pH was monitored in BCECF-loaded cells, and the recovery rates from internal acidification upon the addition of Na⁺-containing Ringer's solution were monitored. Rates were compared between cells pretreated with Zn²⁺ (100 μm) applied in Ringer's solution at the indicated pH or in control cells that were incubated in Zn²⁺-free Ringer's solution at pH 7.4. Representative traces of the pH_i recovery after the NH₄⁺ prepulse paradigm (top panel, for simplicity only the recovery phase after the addition of Na⁺ is shown) and the averaged recovery rates (bottom panel) are shown (n = 3; *, p < 0.05 compared with WT control; #, p < 0.05 compared with D313A control). N.S., not significant.