Characterization of a Functional Bacterial Homologue of Sodium-dependent Neurotransmitter Transporters

Abstract

The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na ؉ -dependent and highly selective. The K m for tryptophan was ϳ145 nM, and tryptophan transport was unchanged in the presence of 100 M concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K I values of 200 and 440 M, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His 6 tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.Transporters responsible for reuptake of neurotransmitters across the plasma membrane of neurons and glia fall into two gene families (1). The majority of small neurotransmitters, including glycine, ␥-aminobutyric acid (GABA), 1 dopamine, norepinephrine, and 5-hydroxytryptamine (5-HT, serotonin), are transported by proteins belonging to the family designated the neurotransmitter:sodium symporter (NSS) family 2.A.22 by Saier (2). Glutamate, however, is transported by a family of mono-and dicarboxylic amino acid transporters, the dicarboxylate/amino acid:cation symporters family (2). Proteins in both families play important roles in brain function as indicated by the profound behavioral effects of drugs that influence their activity, such as cocaine and amphetamines, which interact with amine transporters in the NSS family (3-12), and many antidepressant drugs that inhibit serotonin and norepinephrine transporters (13-17).Among the sequences found to be homologous to the NSS family of transporters are a number of "orphan" transporters, for which no function is known. These orphans include v7-3 (18), NTT4 (19,20), inebriated (21), blot (22), and NTT5 (23), among others. The largest number of orphan sequences in this family is found in prokaryotic organisms. Although these orphan sequences are highly similar to those encoding functional transporters, it is possible that these proteins fulfill other functions. For example, within the ATP-binding cassette family of transporters are the sulfonylurea receptor (24) and the cystic fibrosis transmembrane regulator chloride channel (25). In the dicarboxylate/amino acid:cation symporters neurotransmitter transporter family is EAAT4, a ligand-gated ion channel (26); SGLT3, a member of the sodium:solute symporter (SSS) sugar transporter family, also is not a transporter but rather a glucose-gated ion channel.2 Moreover, some proteins, such as adenylate cyclase (28) and patched (29) also have 12 transmembrane segments but no known transport function. For the orphan transporters in the NSS family, it is important to know if any of the newly discovered prokaryotic sequences actually encode functional transporters.Symbiobacterium thermophilum is a symbiotic thermophile, the growth of which is dependent on co-culture with an associated...

Identification of a chloride ion binding site in Na + /Cl − -dependent transporters

“…In contrast, the prokaryotic tryptophan and tyrosine transporters, stTnaT and fnTyt1 (SI Fig. 5), contain Asp and Ala residues, respectively, at the position corresponding to E290; both of these proteins transport amino acids in a Cl Ϫ -independent manner (15,16).…”

Ancestry and progeny of nutrient amino acid transporters

“…4). In addition several members of bacteria and archaean SNF form a cluster which is labeled as a ''prokaryote NATs'' because these transporters supply essential carbon in several prokaryotes by Na ϩ -driven accumulation of specific amino acids (24). Several metazoan SNF members differ by out-branching points between prokaryote and metazoan NATs, and participating lineages are labeled as a ''metazoan orphan SNF'' because the transport phenotype or other functions of these transporters remain unidentified, except for ''orphan BLOT'' transporters, whose essential role in cell differentiation and epithelial morphogenesis has been inferred from morphological mapping and knockout analysis (25).…”

Transmembrane Domain 8 of the γ-Aminobutyric Acid Transporter GAT-1 Lines a Cytoplasmic Accessibility Pathway into Its Binding Pocket

“…The sodium ions in the binding pocket are both close to the substrate, which is in direct contact, through its carboxyl group, with Na1 (12). In contrast to NSS neurotransmitter transporters, LeuT and other bacterial homologues do not require chloride for transport (12,19,20), and indeed, no chloride was observed in the binding pocket of LeuT (12). Recently, the chloride binding site of the NSS neurotransmitter transporters was identified.…”