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.ATP ͉ chromaffin granule ͉ purinergic signaling ͉ storage and exocytosis ͉ synaptic vesicle V esicular storage and subsequent exocytosis of neurotransmitters is essential for chemical transmission in neurons and endocrine cells. Thus far four distinct classes of transporters are known to participate in the uptake of neurotransmitters into neuronal synaptic vesicles and secretory granules in endocrine cells. These are vesicular monoamine transporters, vesicular acetylcholine transporters, vesicular inhibitory amino acid transporters, and vesicular glutamate transporters (VGLUTs) (1-7). These vesicular transporters mediate the active accumulation of their respective neurotransmitters through an electrochemical gradient of protons across the membrane generated by vacuolar proton-ATPase. ATP is stored in secretory vesicles and subsequently exocytosed, which leads to the various purinergic responses, such as central control of autonomic functions, pain and mechanosensory transduction, neural-glial interactions, control of vessel tone and angiogenesis, and platelet aggregation through purinoceptors, thus establishing its role as a chemical transmitter (8)(9)(10)(11)(12). Because the concentration of nucleotides in the vesicles was maintained at Ϸ0.1-1 M, an active transport mechanisms to accumulate nucleotides has been postulated (8-18). Although evidence increasingly supports the presence of a vesicular ATP transporters in secretory vesicles such as synaptic vesicles and adrenal chromaffin granules (13-19), the protein responsible for the ATP accumulation has not yet been identified.Here, we report the expression and function of human and mouse SLC17A9, an isoform of the SLC17 phosphate transporter family. We present evidence that the SLC17A9 protein acts as a vesicular nucleotide transporter (VNUT) and that it plays an essential role in vesicular storage of ATP in the ATP-secreting cells. Results a...

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…Acinar cells also secrete digestive enzymes and nucleotideconverting enzymes [5,7], and our earlier studies indicated that zymogen granules and fluorescent markers for ATP stores are co-localized [4]. Sawada and coworkers published their discovery of a putative ATP transporter (SLC17A9), named vesicular nucleotide transporter (VNUT), which was expressed in enterochromaffin granules [8]. Soon after, we provided evidence that VNUT is expressed in pancreatic zymogen granules and is engaged in ATP uptake [9].…”

“…The cells were subsequently fixed in 4 % paraformaldehyde in phosphate buffered saline (PBS) for 15 min, treated with 0.2 M TRIS-glycine (pH 7.4) for 15 min, and then rinsed in PBS and permeabilized for 10 min in PBS 0.5 % TritonX-100. The samples were blocked with 10 % bovine serum albumin (BSA) in PBS for 45 min and then incubated with the primary antibodies for VNUT (1:400), which were used in previous studies [8,13], and Rab3D (1:100; Santa Cruz Sc-26559) for 2 h. The slides were then washed in PBS and incubated for 1 h with 1:400 dilution of appropriate secondary antibody conjugated to Alexa 488 or Alexa 568. For nuclear staining, DAPI (4',6-diamidino-2-phenylindole) was used (1:400).…”

“…The two original templates were the VNUT plasmid [8,14] and the Clontech mCherry (from pmCherry-C1 vector) together with plasmid pPAP7160. Plasmid pPAP7160 was generated by homologous recombination in yeast strain PAP1500 [19] between a polymerase chain reaction (PCR) fragment encompassing nucleotides 1-2,580 from pEGFP-N1 (Clontech, USA) and pEMBLyex4 [20].…”

Signal transduction in astrocytes: Localization and release of inorganic polyphosphate

“…Fusions with EGFP or mKate2.7 allow detection of transgene expression via green or red fluorescence, respectively. The VGLUT2 (vesicular glutamate transporter; NM0534271; kind gift from P. Bezzi, Lausanne (Bezzi et al, 2004) is targeted to glutamatergic vesicles in astrocytes, while the mVNUT (vesicular nucleotide transporter; kind gift from T. Miyaji, Japan) delineates putative ATP‐containing vesicles (Sawada et al, 2008). CD63 is a lysosomal membrane protein which localizes to lysosome‐derived exocytotic vesicle‐like structures in astrocytes (CD63 clone BC063173 obtained from ImaGenes, Berlin (Metzelaar et al, 1991).…”

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

“…Exocytotic mechanisms involve ATP storage and Ca 2+ -regulated exocytotic release and SLC17A9 protein has been identified as a vesicular nucleotide transporter (VNUT) that plays a key role in ATP exocytosis [13]. VNUT expression has been detected in different types of cells [13][14][15][16][17][18][19]; however, its presence in corneal and conjunctival epithelial cells remains elusive.…”