Figure 2.

ZnR/GPR39 mediates Zn²⁺-dependent intracellular Ca²⁺ signaling in TAMR cells. (A) Representative Fura-2 fluorescent signal of TAMR cells pre-treated with the Gαq inhibitor, YM-254890 (1 μM, 5 min) or without it (control). At the indicated time, Zn²⁺ (200 μM) was applied. Inset shows the Ca²⁺ response of YM-254890 treated cells to the purinergic agonist ATP (25 μM). (B) Representative Ca²⁺ response of TAMR cells transfected with siGPR39 or a scrambled control (siControl) to Zn²⁺ (200 μM, as indicated, left panel). Right panel (top) shows the response of siGPR39 cells to the purinergic agonist ATP (25 µM). Right panel (bottom) depicts mRNA level of ZnR/GPR39 in siGPR39 and siControl cells (n = 4 coverslips for each condition, in 6 independent experiments, p < 0.05 t-test). (C) TAMR cells were pre-treated with lower concentration of Zn²⁺ for a prolonged time (100 μM for 25 min, 37 °C). Cells were then maintained in Zn²⁺-free Ringer’s solution for 30 min (desensitized) or returned to the incubator for 24 h (recovery, n = 4 coverslips), and subsequently ZnR/GPR39 activation was monitored using the paradigm described in Fig. 1. Representative Ca²⁺ signals in control cells, desensitized cells and cells following recovery are shown. (D) Average rates of initial Ca²⁺ response as determined from A-C. (n = 4 coverslips for each condition, in 6 independent experiments, p < 0.05 t-test). (E) Fluorescent imaging of MCF-7 cells transfected with an mCherry- vector or mCherry-ZnR/GPR39. (F) Representative Fura-2 signals, indicating Ca²⁺ responses, of MCF-7 cells transfected with the mCherry-vector or mCherry-ZnR/GPR39, following application of Zn²⁺ (200 μM, at the indicated time). Inset shows the purinergic response of the mCherry-vector cells, indicating that the metabotropic pathway is intact in these cells. (G) Cells expressing mCherry-vector or mCherry-ZnR/GPR39 were desensitized using the paradigm described in C. After 30 min in Zn²⁺-free Ringer’s solution, Fura-2 loaded cells were imaged and the initial rate of the response to Zn²⁺ (200 μM) was determined. The average of the initial rate of the Zn²⁺-dependent Ca²⁺ response in desensitized cells or controls is shown. (n = 5 coverslips for each condition, in 4 independent experiments, p < 0.05 ANOVA).