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A Na؉ -dependent neutral and cationic amino acid transport system (B 0؉) plays an important role in many cells and tissues; however, the molecular basis for this transport system is still unknown. To identify new transporters, the expressed sequence tag database was queried, and cDNA fragments with sequence similarity to the Na ؉ /Cl ؊ -dependent neurotransmitter transporter family were identified. Based on these sequences, rapid amplification of cDNA ends of human mammary gland cDNA was used to obtain a cDNA of 4.5 kilobases (kb). The open reading frame encodes a 642-amino acid protein named amino acid transporter B 0؉ . Human ATB 0؉(hATB 0؉ ) is a novel member of the Na ؉ /Cl ؊ -dependent neurotransmitter transporter family with the highest sequence similarity to the glycine and proline transporters. Northern blot analysis identified transcripts of ϳ4.5 kb and ϳ2 kb in the lung. Another tissue survey suggests expression in the trachea, salivary gland, mammary gland, stomach, and pituitary gland. Electrophysiology and radiolabeled amino acid uptake measurements were used to functionally characterize the transporter expressed in Xenopus oocytes. hATB 0؉ was found to transport both neutral and cationic amino acids, with the highest affinity for hydrophobic amino acids and the lowest affinity for proline. Amino acid transport was Na ؉ and Cl ؊ -dependent and was attenuated in the presence of 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid, a system B 0؉ inhibitor. These characteristics are consistent with system B 0؉ amino acid transport. Thus, hATB 0؉ is the first cloned B 0؉ amino acid transporter.

This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3 H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3 H]GABA and [ 3 H]tiagabine binding. Voltage jumps, Na ϩ and Cl Ϫ concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na ϩ ions interact with the transporter even in the absence of GABA, and (2) that Cl Ϫ facilitates the binding of Na ϩ . Comparison between the charge movements and the transport-associated current shows that this initial Na ϩ -transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation-treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na ϩ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na ϩ substitutes Li ϩ and Cs ϩ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na ϩ and Cl Ϫ , and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.

No abstract

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.