Divalent cation transport by vesicular nucleotide transporter

Abstract

The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.

FIGURE 1.

Mg²⁺ uptake by VNUT-containing proteoliposomes as assessed by atomic absorption spectrometry. Purified VNUT and reconstituted VNUT were suspended in a reaction mixture containing 100 μm ATP and 1 mm magnesium acetate. Uptake was measured at 2 min after the addition of valinomycin (Val). Mg²⁺ uptake was expressed after subtraction of the value for liposomes without VNUT. Error bars represent means ± S.D. (n = 3–5).

FIGURE 2.

VNUT is responsible for radioactive Ca²⁺ uptake by reconstituted proteoliposomes. A, time course. The assay mixture was incubated with radioactive Ca²⁺ at a final concentration of 1 mm in the presence (●) or absence (○) of 100 μm ATP. The reaction was started by the addition of valinomycin (Val). Ca²⁺ uptake by liposomes lacking VNUT is also shown (□). B, stoichiometry of Ca²⁺ and ATP uptake. Ca²⁺ and ATP uptake was measured in the presence of 100 μm ATP and 1 mm calcium acetate. Error bars represent means ± S.D. (n = 3–5).

FIGURE 3.

Other SLC17 family members do not exhibit Ca²⁺ uptake activity. NPT1, VEAT, and VGLUT2 were purified and reconstituted into liposomes. The valinomycin-evoked Ca²⁺ uptake at 2 min was examined in the presence of 100 μm p-aminohippuric acid for NPT1, 100 μm aspartate for VEAT, and 100 μm glutamate for VGLUT2 and valinomycin (Val). The ATP-dependent Ca²⁺ uptake by VNUT at 2 min is also shown as a positive control. Error bars represent means ± S.D. (n = 3–5).

FIGURE 4.

Effect of Cl⁻ on Ca²⁺ uptake. Ca²⁺ uptake was assayed in the presence of different concentrations of Cl⁻ in the presence (●) or absence (○) of valinomycin (Val). Error bars represent means ± S.D. (n = 3–5).

FIGURE 5.

Effects of inhibitors of VGLUT and various Ca²⁺-transporting proteins on Ca²⁺ uptake. The additions were 1 μm Evans blue, 10 μm DIDS, 200 μm atractyloside, 100 μm ruthenium red, and 200 μm thapsigargin. Ca²⁺ uptake was measured at 2 min after the addition of valinomycin (Val). Control activity (100%) corresponds to 20.7 ± 1.8 nmol/mg. Error bars represent means ± S.D. (n = 3–5).

FIGURE 6.

Effects of divalent cations on Ca²⁺ uptake. A, Ca²⁺ uptake at 2 min was measured in the presence of various cations at 1 mm. Control activity (100%) corresponds to 21.8 ± 2.4 nmol/mg. B, Ca²⁺ uptake was assayed in the presence of different concentrations of magnesium or lanthanum. Ca²⁺ uptake was measured at 2 min after the addition of valinomycin (Val). Error bars represent means ± S.D. (n = 3–5).

FIGURE 7.

Effects of mutations on ATP and Ca²⁺ uptake. The VNUT mutants were purified, and ATP uptake (left panel) and Ca²⁺ uptake (right panel) were measured at 2 min after the addition of valinomycin (Val). Coomassie Brilliant Blue staining of the purified wild-type and mutant VNUT-containing samples is shown in the inset. Error bars represent means ± S.D. (n = 3–5).

FIGURE 8.

Effects of divalent cations on kinetics of nucleotide uptake. The dose dependence of nucleotide uptake in the presence of 5 mm magnesium acetate (○), 5 mm calcium acetate (□), or 1 mm EGTA (●) was measured at 1 min after the addition of valinomycin. The nucleotide transport activities after background subtraction are shown.