Reassessment of the Transport Mechanism of the Human Zinc Transporter SLC39A2

Abstract

The human zinc transporter SLC39A2, also known as ZIP2, was shown to mediate zinc transport that could be inhibited at pH <7.0 and stimulated by HCO₃^-, suggesting a Zn²⁺/HCO₃^- cotransport mechanism [Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564]. In contrast, recent experiments in our laboratory indicated that the functional activity of ZIP2 increases at acidic pH [Franz, M. C., et al. (2014) J. Biomol. Screening 19, 909-916]. The study presented here was therefore designed to reexamine the findings about the pH dependence and to extend the functional characterization of ZIP2. Our current results show that ZIP2-mediated transport is modulated by extracellular pH but independent of the H⁺ driving force. Also, in our experiments, ZIP2-mediated transport is not modulated by extracellular HCO₃^-. Moreover, a high extracellular [K⁺], which induces depolarization, inhibited ZIP2-mediated transport, indicating that the transport mechanism is voltage-dependent. We also show that ZIP2 mediates the uptake of Cd²⁺ ( K_m ∼ 1.57 μM) in a pH-dependent manner ( K_H⁺ ∼ 66 nM). Cd²⁺ transport is inhibited by extracellular [Zn²⁺] (IC₅₀ ∼ 0.32 μM), [Cu²⁺] (IC₅₀ ∼ 1.81 μM), and to a lesser extent [Co²⁺], but not by [Mn²⁺] or [Ba²⁺]. Fe²⁺ is not transported by ZIP2. Accordingly, the substrate selectivity of ZIP2 decreases in the following order: Zn²⁺ > Cd²⁺ ≥ Cu²⁺ > Co²⁺. Altogether, we propose that ZIP2 is a facilitated divalent metal ion transporter that can be modulated by extracellular pH and membrane potential. Given that ZIP2 expression has been reported in acidic environments [Desouki, M. M., et al. (2007) Mol. Cancer 6, 37; Inoue, Y., et al. (2014) J. Biol. Chem. 289, 21451-21462; Tao, Y. T., et al. (2013) Mol. Biol. Rep. 40, 4979-4984], we suggest that the herein described H⁺-mediated regulatory mechanism might be important for determining the velocity and direction of the transport process.

Figure 1.

Effect of extracellular pH and bicarbonate upon ⁶³Zn²⁺ uptake by ZIP2-expressing X. laevis oocytes. Uptake of ⁶³Zn²⁺ in the presence of 100 μM ZnCl₂ by ZIP2- and H₂O-injected X. laevis oocytes was measured (A) at different extracellular pH values (5–8.2) and (B) in the absence (pH 6 and 8.2) and presence of HCO₃⁻ (96 mM) at pH 8.2. Data from three different batches of oocytes were normalized to the mean Zn²⁺ uptake by ZIP2 at pH 6.0 (580 ± 124 to 403 ± 62 pmol oocyte⁻¹ min⁻¹) and are represented as means ± SD (6–26 oocytes).

Figure 2.

Role of bicarbonate and protons in zinc uptake by ZIP2-expressing X. laevis oocytes. Representative trace of intracellular pH (pH_i) changes in response to perfusion of (A) CO₂ (5%) and HCO₃⁻ (33 mM) or (B) ND96 at pH 6.0 in the absence and presence of Zn²⁺ (100 μM). Transport activity of ZIP2 was monitored as the change in pH_i when Zn²⁺ was added and removed extracellularly. (C) Summary of the pH change and pH slope (10⁻⁵ pH unit/s) determined after the perfusion of each of the different media. Results are means ± SD (2–6 oocytes).

Figure 3.

Electrophysiological properties of ZIP2-expressing X. laevis oocytes. (A) Representative trace of the current−voltage relationship under the indicated conditions (V_h = −60 mV; 100 μM Zn²⁺). (B) Average currents recorded under the indicated conditions. For each individual oocyte, the data were normalized to the current recorded at −120 mV in pH 7.5 medium (−159.37 to −38.75 nA). Data from the different oocytes (n = 6) were pooled together and are represented as means ± SD.

Figure 4.

Effect of sodium, chloride, and potassium extracellular concentration on ZIP2 transport activity. (A) Changes in fluorescence intensity of Calcium 5 dye in response to Cd²⁺ perfusion (1 μM) measured in HEK293 cells transiently transfected with DsRed-Express2 and ZIP2 DNA constructs in KB buffer (pH 6.5) in which extracellular Na⁺ or Cl⁻ was replaced with equimolar NMDG, choline⁺, and K⁺ or gluconate salts, respectively. Data from two independent experiments were normalized to the mean ZIP2 activity at pH 6.5 and are represented as means ± SD (n = 8–12). (B) Uptake of ⁶³Zn²⁺ in the presence of 100 μM ZnCl₂ by ZIP2- and H₂O-injected X. laevis oocytes measured in ND96 (pH 6.0) in which extracellular Na⁺ was replaced with equimolar choline⁺ or K⁺. Data from two different batches of oocytes were normalized to the mean Zn²⁺ uptake by ZIP2 at pH 6.0 (403 ± 62 pmol oocyte^–1 min^–1) and are represented as means ± SD (6–12 oocytes). P values establish statistical differences between hZIP2-mediated Zn²⁺ uptake at pH 6.0 and the indicated experimental conditions.

Figure 5.

Effect of potassium on membrane potential in ZIP2 transiently transfected HEK293 cells. Representative images of (A) intact or (D) dialyzed cells before and after the treatment with Cd²⁺ (10 μM) in the presence (control) or absence of extracellular 120 mM K⁺. Representative traces of Cd²⁺-flux-induced changes in fluorescence intensity in (B) intact and (E) clamped cells in the presence and absence of 120 mM K⁺. Fluorescence intensity changes were measured as ΔF/F₀ (where F₀ is the signal before application of Cd²⁺). Data from three and five independent experiments for intact (n = 38–41) and dialyzed (n = 8–10) cells, respectively, are represented as means ± SD.

Figure 6.

Kinetics and pH dependence of the Cd²⁺ transport measured in ZIP2 or DsRed-Express2 (empty vector) transiently transfected HEK293 cells. Representative experiments showing the changes in fluorescence intensity of Calcium 5 dye in response to the perfusion of different Cd²⁺ concentrations (0.1–10 μM) at extracellular pH 6.5 (A) or at different extracellular pH values (6.5–8.2) in the presence of a saturating concentration of Cd²⁺ (10 μM) (B). To determine the kinetics (C) and pH dependence (D) of Cd²⁺ transport, the AUC for each single trace was calculated. Data from three independent experiments were normalized to the mean Cd²⁺ uptake by ZIP2 at pH 6.5 (337.22 ± 34 to 890 ± 22 AUC) and collected and are represented as means ± SD (n = 7–28). Kinetic parameters were obtained by fitting the data points to the Michaelis−Menten equation (solid lines).

Figure 7.

Divalent cation selectivity of ZIP2 in transiently transfected HEK293 cells. (A) Changes in fluorescence intensity of Calcium 5 dye in response to Cd²⁺ perfusion (1 μM) measured in the presence of the indicated divalent cations (50 μM) at pH 6.5. Data from two independent experiments were normalized to the mean ZIP2 activity at pH 6.5 in the absence of divalent cations other than Cd²⁺ (1 μM) and are represented as means ± SD (n = 5–8). P values establish statistical differences between ZIP2-mediated Cd²⁺ uptake in the absence and presence of the indicated divalent metals. (B) Uptake of ⁵⁵Fe²⁺ in the presence of 1 μM FeCl₂ and 100 μM ascorbic acid by HEK293 cells transiently transfected with ZIP2, DsRed-Express2 (empty vector) and DMT1 DNA constructs at extracellular pH 5.5. Data from two independent experiments were normalized to the mean ZIP2 ⁵⁵Fe²⁺ transport (0.09 ± 0.01 to 0.15 ± 0.02 pmol min⁻¹) and are represented as means ± SD (n = 19−48). Inhibition of the ZIP2-mediated Cd²⁺ transport (1 μM) by increasing extracellular [Zn²⁺] (C) and [Cu²⁺] (D). Data from two independent experiments were normalized to the mean ZIP2 activity at pH 6.5 in the absence of divalent cations other than Cd²⁺ and are represented as means ± SD (n = 7). Inhibitory kinetics were obtained by fitting the data points to a four-parameter sigmoidal equation (solid lines).