Identification of a vesicular nucleotide transporter

Abstract

ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.

Fig. 1.

Phylogenetic tree of SLC17 family (A) and putative secondary structure of SLC17A9 protein (B).

Fig. 2.

Expression of SLC17A9 and its association with chromaffin granules. (A) Northern blotting showing expression of SLC17A9 in human and mouse brain and adrenal gland. Expression of G3PDH is also shown as a control. (B) Immunohistochemical localization of SLC17A9 protein in mouse adrenal gland. (Inset) Shown is control staining with preabsorbed antibodies. C, cortex; M, medulla. (C) Immunoelectronmicroscopy showing that SLC17A9 proteins (gold particles) are associated with chromaffin granules. (Inset) Shown is background labeling with control serum. Mit, mitochondrion. (D) Western blot indicates the presence of a SLC17A9 counterpart in the membrane of bovine adrenal chromaffin granule with a relative mobility of 61 kDa. The preabsorbed antibodies did not bind to the protein. The positions of marker proteins are indicated on the left. (Scale bars: B, 10 μm; C, 100 nm.)

Fig. 3.

Purification and reconstitution of SLC17A9 protein. (A) Purification. (Left) Purified fraction (7 μg protein) was analyzed in an 11% PAGE in the presence of SDS and visualized by Coomassie brilliant blue staining. (Center) A duplicate gel was analyzed by Western blotting with anti-SLC17A9 antibodies. (Right) Labeling of SLC17A9 protein upon UV light illumination with [α-³²P]ATP. (B) Formation of Δψ (positive inside) was measured by oxonol-V fluorescence quenching. Proteoliposomes (0.5 μg protein) or liposomes (20 μg lipid) containing trapped Na⁺ were suspended in buffer containing 0.15 M K-acetate plus 4 mM KCl and 1 μM oxonol-V, and fluorescence quenching was measured. Final concentration of valinomycin and carbonylcyanide m-chlorophenylhydrazone (CCCP) were added at 1 μM. (C) Time course of [α-³²P]ATP uptake. Na⁺-trapped proteoliposomes or liposomes were suspended as above in the presence or absence of valinomycin. Upon the addition of [α-³²P]ATP, samples were taken at the indicated times, and radioactivity taken up by the proteoliposomes was counted. (Inset) Dose dependence of ATP uptake.

Fig. 4.

Characterization of nucleotides transport by SLC17A9 protein. (A) The effect of divalent cations on the [α-³²P]ATP uptake in the absence or presence of 2 mM Mg²⁺ or Ca²⁺ or 0.5 mM EGTA. (B) Dependence of [α-³²P]ATP uptake on [Cl⁻]. The uptake was measured after 2 min. Part of the potassium acetate in the reaction mixture was replaced with the indicated concentration of KCl. The magnitude of Δψ (oxonol V-fluorescence quenching) was little affected under the assay conditions used. (C) [α-³²P]ATP uptake in the presence of various nucleotides at 1 mM. (D) Uptake of radiolabeled GTP or ADP transport in the presence or absence of valinomycin. When indicated, DIDS at 2 μM was included. (E–G) The effect of DIDS, Evans blue, and atractyloside on [α-³²P]ATP uptake in the presence or absence of Mg-acetate. Atractyloside was included at 200 μM. ∗, P <0.1; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Fig. 5.

The impact of rat SLC17A9 siRNA on the endogenous expression of SLC17A9 in in PC12 cells was compared with cells treated with scrambled siRNA (control). (A) Double-labeling immunohistochemistry indicated that SLC17A9 protein was colocalized at least in part with synaptotagmin, a marker of secretory granule, but not with synaptophysin, a marker of synaptic-like microvesicles in cultured PC12 cells. (B and C) Both SLC17A9 mRNA levels (B), as determined by real-time PCR, and the KCl-dependent secretion of ATP after 30 min (C) were assessed. n = 9 for three independent experiments. ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Fig. 6.

Schematic presentation of purinergic chemical transmission. VNUT is present in secretory vesicles and is responsible for vesicular storage of nucleotides. The accumulated nucleotides, in particular ATP, are exocytosed upon stimulation and bind to purinoceptors at the surface of target cells, which then triggers intracellular signal transmission.