title: 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.
authors: Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan.
authors: Takaaki
authors: T
authors: Miyaji
authors: Keisuke
authors: K
authors: Sawada
authors: Hiroshi
authors: H
authors: Omote
authors: Yoshinori
authors: Y
authors: Moriyama
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism*; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D009711:Nucleotides / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000378:metabolism; D050481:Vesicular Neurotransmitter Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D050491:Glutamate Plasma Membrane Transport Proteins; D009711:Nucleotides; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D050481:Vesicular Neurotransmitter Transport Proteins; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium