Divalent Cation Transport by Vesicular Nucleotide Transporter

• Published in: The Journal of biological chemistry Vol. 286; no. 50; pp. 42881 - 42887
• Main Authors: Miyaji, Takaaki, Sawada, Keisuke, Omote, Hiroshi, Moriyama, Yoshinori
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.12.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - metabolism; Animals; ATP; Biological Transport; Calcium; Calcium - metabolism; Cations, Divalent - metabolism; Chromaffin Granule; Divalent Cation Transport; Glutamate Plasma Membrane Transport Proteins - genetics; Glutamate Plasma Membrane Transport Proteins - metabolism; Humans; Kinetics; Magnesium; Magnesium - metabolism; Membrane Biology; Mice; Neurotransmitter Transport; Nucleotides - metabolism; PC12 Cells; Rats; Reconstitution of Membrane Transporters; Sodium-Phosphate Cotransporter Proteins, Type III - metabolism; Substrate Specificity; Transport; Vesicular Neurotransmitter Transport Proteins - genetics; Vesicular Neurotransmitter Transport Proteins - metabolism; Vesicular Nucleotide Transporter; Calcium; Divalent Cation Transport; Vesicular Nucleotide Transporter; Substrate Specificity; Neurotransmitter Transport; Magnesium; Reconstitution of Membrane Transporters; Transport; Chromaffin Granule; ATP
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.277269

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 Mg2+ and Ca2+, 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 Mg2+ when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca2+ upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca2+ 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 Ca2+. Ca2+ uptake was also inhibited in the presence of various divalent cations such as Mg2+. Kinetic analysis indicated that Ca2+ or Mg2+ did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg2+ 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. Background: The vesicular nucleotide transporter (VNUT) transports nucleotides in the presence of Mg2+. Results: VNUT transports divalent cations in a membrane potential- and nucleotide-dependent manner. Conclusion: VNUT transports divalent cations as a nucleotide complex. Significance: VNUT functions as a divalent cation importer in secretory vesicles.