The C-terminal domain of the neutral amino acid transporter SNAT2 regulates transport activity through voltage-dependent processes

SNAT (sodium-coupled neutral amino acid transporter) 2 belongs to the SLC38 (solute carrier 38) family of solute transporters. Transport of one amino acid molecule into the cell is driven by the co-transport of one Na(+) ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. In the present paper, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of this deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (<0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were normal in the truncated transporter. SNAT2(Del C-ter) (13 residues deleted from the C-terminus) showed 3-fold higher apparent affinity for alanine, and 2-fold higher Na(+) affinity compared with wild-type SNAT2, suggesting that the C-terminus is not required for high-affinity substrate and Na(+) interaction with SNAT2. The pH sensitivity of amino acid transport was retained partially after the truncation. In contrast with the truncation after TM (transmembrane domain) 11, the deletion of TM11 resulted in an inactive transporter, most probably due to a defect in cell surface expression. Taken together, the results demonstrate that the C-terminal domain of SNAT2 is an important voltage regulator that is required for a normal amino acid translocation process at physiological membrane potentials. However, the C-terminus appears not to be involved in the regulation of membrane expression.     

Membrane topological structure of neutral system N/A amino acid transporter 4 (SNAT4) protein

Members of system N/A amino acid transporter (SNAT) family mediate transport of neutral amino acids, including l-alanine, l-glutamine, and l-histidine, across the plasma membrane and are involved in a variety of cellular functions. By using chemical labeling, glycosylation, immunofluorescence combined with molecular modeling approaches, we resolved the membrane topological structure of SNAT4, a transporter expressed predominantly in liver. To analyze the orientation using the chemical labeling and biotinylation approach, the "Cys-null" mutant of SNAT4 was first generated by mutating all five endogenous cysteine residues. Based on predicted topological structures, a single cysteine residue was introduced individually into all possible nontransmembrane domains of the Cys-null mutant. The cells expressing these mutants were labeled with N-biotinylaminoethyl methanethiosulfonate, a membrane-impermeable cysteine-directed reagent. We mapped the orientations of N- and C-terminal domains. There are three extracellular loop domains, and among them, the second loop domain is the largest that spans from amino acid residue ～242 to ～335. The orientation of this domain was further confirmed by the identification of two N-glycosylated residues, Asn-260 and Asn-264. Together, we showed that SNAT4 contains 10 transmembrane domains with extracellular N and C termini and a large N-glycosylated, extracellular loop domain. This is the first report concerning membrane topological structure of mammalian SNAT transporters, which will provide important implications for our understanding of structure-function of the members in this amino acid transporter family.     

Highly conserved asparagine 82 controls the interaction of Na+ with the sodium-coupled neutral amino acid transporter SNAT2

The neutral amino acid transporter 2 (SNAT2), which belongs to the SLC38 family of solute transporters, couples the transport of amino acid to the cotransport of one Na(+) ion into the cell. Several polar amino acids are highly conserved within the SLC38 family. Here, we mutated three of these conserved amino acids, Asn(82) in the predicted transmembrane domain 1 (TMD1), Tyr(337) in TMD7, and Arg(374) in TMD8; and we studied the functional consequences of these modifications. The mutation of N82A virtually eliminated the alanine-induced transport current, as well as amino acid uptake by SNAT2. In contrast, the mutations Y337A and R374Q did not abolish amino acid transport. The K(m) of SNAT2 for its interaction with Na(+), K(Na(+)), was dramatically reduced by the N82A mutation, whereas the more conservative mutation N82S resulted in a K(Na(+)) that was in between SNAT2(N82A) and SNAT2(WT). These results were interpreted as a reduction of Na(+) affinity caused by the Asn(82) mutations, suggesting that these mutations interfere with the interaction of SNAT2 with the sodium ion. As a consequence of this dramatic reduction in Na(+) affinity, the apparent K(m) of SNAT2(N82A) for alanine was increased 27-fold compared with that of SNAT2(WT). Our results demonstrate a direct or indirect involvement of Asn(82) in Na(+) coordination by SNAT2. Therefore, we predict that TMD1 is crucial for the function of SLC38 transporters and that of related families.     

Impact of forskolin and amino acid depletion upon System A activity and SNAT expression in BeWo cells

Amino acid transport System A (SysA) plays an important role in mediating the transplacental transfer of neutral amino acids from mother to fetus. Given that prior work has demonstrated that SysA activity is regulated, both over gestation and in response to dietary restriction during pregnancy, we examined the response of SysA activity and sodium-dependent neutral amino acid transporter (SNAT; responsible for SysA activity) expression to cAMP analogues and amino acid deprivation in BeWo cells, an accepted model of placental syncytia. SysA activity was unaffected by forskolin, a cAMP agonist, at 48 and 72 h. Amino acid depletion was associated with an up-regulation of SysA activity, largely mediated through an enhancement of SNAT2 (Slc38a2) expression at both the protein and mRNA level. SNAT1 (Slc38a1) expression did not change in response to amino acid depletion, while SNAT4 (Slc38a4) could not be detected. In summary, SysA activity in BeWo cells responds to amino acid depletion through the differential regulation of SNAT subtypes.     

The sodium-coupled neutral amino acid transporter SNAT2 mediates an anion leak conductance that is differentially inhibited by transported substrates

The sodium-coupled neutral amino acid transporter SNAT2 mediates cellular uptake of glutamine and other small, neutral amino acids. Here, we report the existence of a leak anion pathway associated with SNAT2. The leak anion conductance was increased by, but did not require the presence of, extracellular sodium. The transported substrates L-alanine, L-glutamine, and alpha-(methylamino)isobutyrate inhibited the anion leak conductance, each with different potency. A transporter with the mutation H-304A did not catalyze alanine transport but still catalyzed anion leak current, demonstrating that substrate transport is not required for anion current inhibition. Both the substrate and Na+ were able to bind to the SNAT2H-304A transporter normally. The selectivity sequence of the SNAT2H-304A anion conductance was SCN->NO3->I->Br->Cl->Mes-. Anion flux mediated by the more hydrophobic anion SCN- was not saturable, whereas nitrate flux demonstrated saturation kinetics with an apparent Km of 29 mM. SNAT2, which belongs to the SLC38 family of transporters, has to be added to the growing number of secondary, Na+-coupled transporters catalyzing substrate-gated or leak anion conductances. Therefore, we can speculate that such anion-conducting pathways are general features of Na+-transporting systems.     

Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative

Na+-Dependent transmembrane transport of small neutral amino acids, such as glutamine and alanine, is mediated, among others, by the neutral amino acid transporters of the solute carrier 1 [SLC1, alanine serine cysteine transporter 1 (ASCT1), and ASCT2] and SLC38 families [sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4]. Many mechanistic aspects of amino acid transport by these systems are not well-understood. Here, we describe a new photolabile alanine derivative based on protection of alanine with the 4-methoxy-7-nitroindolinyl (MNI) caging group, which we use for pre-steady-state kinetic analysis of alanine transport by ASCT2, SNAT1, and SNAT2. MNI-alanine has favorable photochemical properties and is stable in aqueous solution. It is also inert with respect to the transport systems studied. Photolytic release of free alanine results in the generation of significant transient current components in HEK293 cells expressing the ASCT2, SNAT1, and SNAT2 proteins. In ASCT2, these currents show biphasic decay with time constants, tau, in the 1-30 ms time range. They are fully inhibited in the absence of extracellular Na+, demonstrating that Na+ binding to the transporter is necessary for induction of the alanine-mediated current. For SNAT1, these transient currents differ in their time course (tau = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (tau approximately 4-5 ms), indicating that they are associated with a fast, previously undetected, electrogenic partial reaction in the SNAT1 transport cycle. The implications of these results for the mechanisms of transmembrane transport of alanine are discussed. The new caged alanine derivative will provide a useful tool for future, more detailed studies of neutral amino acid transport.     

SNAT4 isoform of system A amino acid transporter is expressed in human placenta

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.     

Characterization and Regulation of the Amino Acid Transporter SNAT2 in the Small Intestine of Piglets

The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine.     

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na⁺ into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT_Aa and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na⁺ binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na⁺ binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na⁺ binding to SNAT2, but also dramatically lowers the Na⁺ affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na⁺ binding and which is also expected to form part of the Na⁺ binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na⁺ affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.The sodium-coupled neutral amino acid transporter, SNAT2,² belongs to the SLC38 gene family of solute carrier proteins (1). Together with SNAT1 and -4 (2), it is believed to mediate Na⁺-dependent amino acid transport activity that was classically assigned to System A transporters (3–8). In addition to SNAT1 and -2, the SLC38 family has four other known members, two of which predominantly mediate glutamine transport (SNAT3 and -5, System N (9–11)). SNAT2 is widely expressed in mammalian tissue (1, 7), but it may play a particularly critical role in the brain (12), where it may help shuttle glutamine from astrocytes to neurons via the glutamate-glutamine cycle (1). This process is essential for recycling the neurotransmitter glutamate (13). However, the exact contribution of SNAT2 to the glutamate-glutamine cycle is still controversially discussed (14).Despite this physiological importance, surprisingly little is known about the functional properties and the structural basis of amino acid transport by the SLC38 proteins. Although hydropathy analysis predicts 11 transmembrane helices (TMs), with an intracellular N terminus and an extracellular C terminus (1), it is not clear whether the transporters belong to a large superfamily of transporters, of which members have been characterized structurally through x-ray crystallography. At present, sequence homology has only been established with transporters of the mammalian SLC32 and SLC36 families as well as with the more distantly related plant auxin carriers and the bacterial amino acid-polyamine-organocation (APC) family (15, 16). High resolution crystal structures are not available for any of the transporters from these families, although low resolution projection structures were recently reported for the APC family members AdiC (17) and SteT (18). However, these structures do not allow the assignment of transmembrane helices. Thus, it remains unknown whether the SLC38 fold is similar to established transport protein folds, although homology to the major facilitator superfamily seems unlikely.We have recently identified a conserved amino acid residue in SNAT2, Asn-82, which is involved in controlling the Na⁺ affinity of the transporter (19). Interestingly, Asn-82 is localized in the predicted TM1 of SNAT2. This first transmembrane helix was recently found to contribute ligands to a Na⁺ binding site in several bacterial transporters, which are related to the SLC5 (sodium glucose symporter) and SLC6 (sodium- and chloride-dependent neurotransmitter transporter) family members (20–22), which also comprises bacterial members (23, 24). Although sequence similarity with SLC5 and -6 is not detectable, SLC38 may be a member of a possibly very large superfamily with the same general fold, which also contains many amino acid transport proteins.Here, we used a homology modeling approach based on profile-based sequence alignment (25, 26). A search against sequences deposited in the Protein Data Bank (PDB (27)) revealed that the transporters with the highest likelihood to share an analogous fold are a leucine transporter from Aquifex aeolicus, LeuT_Aa, and a homologous hydantoin transporter from Microbacterium liquefaciens, Mhp1. We established a homology model based on these structures, which predicts Asn-82 to be part of a Na⁺ binding site. Furthermore, another conserved hydrophilic amino acid residue in TM8, Thr-384, was predicted to be near this cation binding site. When Thr-384 was mutated to alanine, a dramatic loss of the affinity of SNAT2 for Na⁺ was observed, whereas mutations to other sites that were spatially removed from the predicted Na⁺ binding site had little or no effect on Na⁺ affinity. We hypothesize that the SLC38 family is a member of a large superfamily of cation/organic substrate transporters which includes the mammalian SLC5 and -6 proteins and which has a conserved cation binding site formed by TMs 1 and 8.     

ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space

Mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a potentially cleavable signal sequence in this 60-residue extension. We investigated the functional role of this extension and found that it was required for stable protein expression of transporter constructs containing any downstream transmembrane domains. The extension directed transporter translocation across the ER membrane with an orientation that resulted in glycosylation of amino acids immediately distal to the signal sequence. Neither the native signal sequence nor a green fluorescent protein tag, fused at the amino terminus, was cleaved from ABCA1. The green fluorescent protein fusion protein had efflux activity comparable with wild type ABCA1 and demonstrated a predominantly plasma membrane distribution in transfected cells. These data establish a requirement for the upstream 60 amino acids of ABCA1. This region contains an uncleaved signal anchor sequence that positions the amino terminus in a type II orientation leading to the extracellular presentation of an approximately 600-amino acid loop in which loss-of-function mutations cluster in Tangier disease patients.     

The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells

There is an increasing appreciation that amino acids can act as signaling molecules in the regulation of cellular processes through modulation of intracellular cell signaling pathways. In culture, embryonic stem (ES) cells can be differentiated to a second, pluripotent cell population, early primitive ectoderm-like cells in response to biological activities within the conditioned medium MEDII. The amino acid l-proline has been identified as a component of MEDII required for ES cell differentiation. Here, we define the primary l-proline transporter on ES and early primitive ectoderm-like cells as sodium-coupled neutral amino acid transporter 2 (SNAT2). SNAT2 uptake of l-proline can be inhibited by the addition of millimolar concentrations of other substrates. The addition of excess amino acids was used to regulate the uptake of l-proline by ES cells, and the effect on differentiation was analyzed. The ability of SNAT2 substrates, but not other amino acids, to prevent changes in morphology, gene expression, and differentiation kinetics suggested that l-proline uptake through SNAT2 was required for ES cell differentiation. These data reveal an unexpected role for amino acid uptake and the amino acid transporter SNAT2 in regulation of pluripotent cells in culture and provides a number of specific, inexpensive, and nontoxic culture additives with the potential to improve the quality of ES cell culture.     

Membrane topology of Bves/Pop1A,a cell adhesion molecule that displays dynamic changes in cellular distribution during development

We investigated the membrane topology of Bves/Pop1A as a foundation to dissect the molecular basis and function of Bves/Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/Pop1A during coronary vessel development.     

Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland

Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.     

Abundance of amino acid transporters involved in mTORC1 activation in skeletal muscle of neonatal pigs is developmentally regulated

Previously we demonstrated that the insulin- and amino acid-induced activation of the mammalian target of rapamycin complex 1 (mTORC1) is developmentally regulated in neonatal pigs. Recent studies have indicated that members of the System A transporter (SNAT2), the System N transporter (SNAT3), the System L transporters (LAT1 and LAT2), and the proton-assisted amino acid transporters (PAT1 and PAT2) have crucial roles in the activation of mTORC1 and that the abundance of amino acid transporters is positively correlated with their activation. This study aimed to determine the effect of the post-prandial rise in insulin and amino acids on the abundance or activation of SNAT2, SNAT3, LAT1, LAT2, PAT1, and PAT2 and whether the response is modified by development. Overnight fasted 6- and 26-day-old pigs were infused for 2 h with saline (Control) or with insulin or amino acids to achieve fed levels while amino acids or insulin, respectively, as well as glucose were maintained at fasting levels. The abundance of SNAT2, SNAT3, LAT1, LAT2, PAT1, and PAT2 was higher in muscle of 6- compared with 26-day-old pigs. The abundance of the PAT2–mTOR complex was greater in 6- than in 26-day-old pigs, consistent with the higher activation of mTORC1. Neither insulin nor amino acids altered amino acid transporter or PAT2–mTOR complex abundance. In conclusion, the amino acid transporters, SNAT 2/3, LAT 1/2, and PAT1/2, likely have important roles in the enhanced amino acid-induced activation of mTORC1 in skeletal muscle of the neonate.     

Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.     

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.