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...

Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl−. Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl− acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl− at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses, and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity.

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F o F 1 -ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (⌬)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for ϳ4 mM Cl ؊ , was sensitive to Evans blue, but was insensitive to D,L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg 184 , His 128 , and Glu 191 showed a dramatic loss in L-glutamate transport activity, whereas Na ؉ -dependent inorganic phosphate (P i ) uptake remained comparable to that of the wild type. Furthermore, P i transport did not require Cl ؊ and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a ⌬-dependent L-glutamate uptake and a Na ؉ -dependent P i uptake.Vesicular storage and subsequent exocytosis of L-glutamate is the major pathway for excitatory signal transmission in the central nervous system (1-3). Vesicular glutamate transporters (VGLUTs) 2 are essential for the vesicular storage of L-glutamate through active transport of L-glutamate into synaptic vesicles at the expense of ⌬H ϩ established by vacuolar H ϩ -ATPase (V-ATPase) (1). There are three isoforms of VGLUT, denoted VGLUT1, VGLUT2, and VGLUT3 on the basis of the order of their discovery (2, 4 -6). VGLUT1 and VGLUT2 show a complementary expression pattern in essentially all known glutamatergic neurons, suggesting that the two VGLUTs are involved in glutamatergic neurotransmission (7-9). In fact, VGLUT1 knock-out mice exhibit a loss of secretion of L-glutamate and glutamatergic neurotransmission in neurons that normally express VGLUT1 (4, 10). In contrast, VGLUT3 is expressed in neurons that are usually classified as non-glutamatergic neurons and astrocytes suggesting the involvement of VGLUT3 in a novel mode of L-glutamate signaling (11-13). VGLUTs are also expressed in peripheral nonneuronal cells, associated with a wide variety of secretory vesicles and are responsible for glutamate-mediated regulation in various cellular processes (5).VGLUTs belong to the SLC17/type I anion transport family, one of the major facilitator superfamilies (MFS), and are not related to other neurotransmitter transporters such as vesicular acetylcholine transporter and vesicular monoamine transporter (2, 14). VGLUT exhibits unique transport properties when compared with other vesicular neurotransmitter transporters. For one, VGLUT is activated by low concentrations of Cl Ϫ (ϳ4 mM) through a putative Cl Ϫ binding site (15-18). Furthermore, VGLUT requires membrane potential (positive inside) as a driving...

Summary In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of a ~65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.

Glutamate plays essential roles in chemical transmission as a major excitatory neurotransmitter. The accumulation of glutamate in secretory vesicles is mediated by vesicular glutamate transporters (VGLUTs) that together with the driving electrochemical gradient of proteins influence the subsequent quantum release of glutamate and the function of higher-order neurons. The vesicular content of glutamate is well correlated with membrane potential (Δψ), which suggests that Δψ determines the vesicular glutamate concentration. The transport of glutamate into secretory vesicles is highly dependent on Cl(-). This anion stimulates glutamate transport but is inhibitory at higher concentrations. Accumulating evidence indicates that Cl(-) regulates glutamate transport through control of VGLUT activity and the H(+) electrochemical gradient. Recently, a comprehensive study demonstrated that Cl(-) regulation of VGLUT is competitively inhibited by metabolic intermediates such as ketone bodies. It also showed that ketone bodies are effective in controlling epilepsy. These results suggest a correlation between metabolic state and higher-order brain function. We propose a novel function for Cl(-) as a fundamental regulator for signal transmission.