Synaptic Vesicle Protein NTT4/XT1 (SLC6A17) Catalyzes Na+-coupled Neutral Amino Acid Transport

Abstract

The SLC6 family of structurally related, Na ؉ -dependent transporter proteins is responsible for presynaptic reuptake of the majority of neurotransmitters. Within this family are a number of orphan transporters, including NTT4/XT1 (SLC6A17), a protein first identified over 15 years ago. NTT4/XT1 is expressed exclusively in the nervous system and specifically on synaptic vesicles in glutamatergic and some GABAergic neurons. Despite extensive efforts by a number of groups, no substrate has been reported for NTT4/XT1. Here we use a combination of molecular manipulations to increase expression of the NTT4/XT1 protein at the plasma membrane and to directly demonstrate that it catalyzes neutral amino acid transport. The substrate profile of the NTT4/XT1-dependent activity is similar to that of the closely related B 0 AT2/SBAT1 (SLC6A15), including a submillimolar apparent affinity for proline and leucine and a low millimolar apparent affinity for glutamine. The transport activity is Na ؉ -dependent and Cl ؊ -independent and is inhibited by low pH as is SLC6A15, suggesting redundant roles for these proteins. This characterization of NTT4/XT1 offers important insights into neurotransmitter metabolism as well as the mechanistic differences among the structurally related, but functionally divergent, SLC6 proteins.Synaptic transmission places a great metabolic burden upon neurons. The thousands of neurotransmitter molecules released with fusion of each synaptic vesicle must be replenished. The most direct mechanism of compensating for exocytotic release is presynaptic reuptake of the neurotransmitter through plasma membrane transporters. Such a reuptake mechanism is used by several types of neurons, including those that release GABA, 2 glycine, and monoamines. The fates of synaptically released acetylcholine and glutamate are more complex; acetylcholine is hydrolyzed to acetate and choline in the synaptic cleft, and synaptically released glutamate is readily taken up by astrocytes, precluding direct neuronal reuptake of these neurotransmitters. Although acetylcholine is hydrolyzed in the synaptic cleft, a presynaptic transport system for the choline portion of the neurotransmitter is expressed in cholinergic neurons (1). Glutamate cleared from the synapse by astrocytes is rapidly converted to glutamine and shuttled back into neurons for reconversion to glutamate (2). Despite the efficiency of astrocytic glutamate uptake, it is estimated that at least 25% of the synaptically released glutamate exits this glutamate-glutamine cycle (3), suggesting that glutamatergic neurons must rely on additional transport mechanisms for uptake of neurotransmitter precursors.The SLC6 family, a group of structurally related, Na ϩ -dependent transporter proteins, is responsible for presynaptic reuptake of the majority of neurotransmitters (4, 5). These transporters can be subdivided into the following groups on the basis of structure: 1) a monoamine transporter group (DAT, NET, and SERT); 2) a GABA transporter group that includes GAT1-3 as wel...

Proteome Analysis and Conditional Deletion of the EAAT2 Glutamate Transporter Provide Evidence against a Role of EAAT2 in Pancreatic Insulin Secretion in Mice

“…ZnT8, which is indispensable for zinc-insulin crystal formation (64). The only vesicular transporter detected was Slc6a17 (RxT; NTT4) which transports proline, glycine, leucine, and alanine (55,56), but it is not known in which of the islet cell types it is expressed.…”

A SLC6 transporter of the novel B0,- system aids in absorption and detection of nutrient amino acids in Caenorhabditis elegans

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“…These properties identify a new transport system, which we named here as B 0,-, following the generally accepted convention for amino acid transport systems (Bannai et al, 1984;Christensen et al, 1994). Recent cloning of several orphan transporters from the mammalian SLC6 family revealed the molecular identity of the B 0 system, which currently incorporates four characterized orthologous groups identified as B 0 AT1 (SLC6A19) (Romeo et al, 2005), B 0 AT2 (SLC6A15) (Takanaga et al, 2005b;Bröer et al, 2006), B 0 AT3 (SLC6A18) (Singer et al, 2009) and NTT4 (SLC6A17) (Zaia and Reimer, 2009) members. Combined, these transporters represent 80% of the NAT subfamily in mammals.…”

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