Expression of glutamine transporter Slc38a3 (SNAT3) during acidosis is mediated by a different mechanism than tissue-specific expression.

BACKGROUND: Despite homeostatic pH regulation, systemic and cellular pH changes take place and strongly influence metabolic processes. Transcription of the glutamine transporter SNAT3 (Slc38a3) for instance is highly up-regulated in the kidney during metabolic acidosis to provide glutamine for ammonia production. METHODS: Slc38a3 promoter activity and messenger RNA stability were measured in cultured cells in response to different extracellular pH values. RESULTS: Up-regulation of SNAT3 mRNA was mediated both by the stabilization of its mRNA and by the up-regulation of gene transcription. Stabilisation of the mRNA involved a pH-response element, while enhanced transcription made use of a second pH-sensitive Sp1 binding site in addition to a constitutive Sp1 binding site. Transcriptional regulation dominated the early response to acidosis, while mRNA stability was more important for chronic adaptation. Tissue-specific expression of SNAT3, by contrast, appeared to be controlled by promoter methylation and histone modifications. CONCLUSIONS: Regulation of SNAT3 gene expression by extracellular pH involves post-transcriptional and transcriptional mechanisms, the latter being distinct from the mechanisms that control the tissue-specific expression of the gene.

Enterocyte-specific regulation of the apical nutrient transporter SLC6A19 (B(0)AT1) by transcriptional and epigenetic networks.

Enterocytes are specialized to absorb nutrients from the lumen of the small intestine by expressing a select set of genes to maximize the uptake of nutrients. They develop from stem cells in the crypt and differentiate into mature enterocytes while moving along the crypt-villus axis. Using the Slc6a19 gene as an example, encoding the neutral amino acid transporter B(0)AT1, we studied regulation of the gene by transcription factors and epigenetic factors in the intestine. To investigate this question, we used a fractionation method to separate mature enterocytes from crypt cells and analyzed gene expression. Transcription factors HNF1a and HNF4a activate transcription of the Slc6a19 gene in villus enterocytes, whereas high levels of SOX9 repress expression in the crypts. CpG dinucleotides in the proximal promoter were highly methylated in the crypt and fully de-methylated in the villus. Furthermore, histone modification H3K27Ac, indicating an active promoter, was prevalent in villus cells but barely detectable in crypt cells. The results suggest that Slc6a19 expression in the intestine is regulated at three different levels involving promoter methylation, histone modification, and opposing transcription factors.

Rapid downregulation of the rat glutamine transporter SNAT3 by a caveolin-dependent trafficking mechanism in Xenopus laevis oocytes.

The glutamine transporter SNAT3 is involved in the uptake and release of glutamine in the brain, liver, and kidney. Substrate transport is accompanied by Na(+) cotransport and H(+) antiport. In this study, treatment of Xenopus laevis oocytes expressing rat SNAT3 with the phorbol ester PMA resulted in a rapid downregulation of glutamine uptake in less than 20 min. PMA treatment of oocytes coexpressing SNAT3 and the monocarboxylate transporter MCT1 reduced SNAT3 activity only, demonstrating the specificity of the regulatory mechanism. Single or combined mutations of seven putative phosphorylation sites in the SNAT3 sequence did not affect the regulation of SNAT3 by PMA. Expression of an EGFP-SNAT3 fusion protein in oocytes established that the downregulation was caused by the retrieval of the transporter from the plasma membrane. Coexpression of SNAT3 with dominant-negative mutants of dynamin or caveolin revealed that SNAT3 trafficking occurs in a dynamin-independent manner and is influenced by caveolin. Although system N activity was not affected by PMA in cultured astrocytes, a downregulation was observed in HepG2 cells.

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20).

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na(+)-binding sites and a putative Cl(-)-binding site. Mutation of all residues in the two proposed Na(+)-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na(+)-binding site 1) and mutants S20A, S20G, S20G/G405S (Na(+)-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na(+)-binding site 1 caused a decrease of the Na(+)-K(0.5), whereas mutations of site 2 increased the K(0.5). Mutation of Na(+)-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates (36)Cl(-) demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na(+)/1Cl(-)-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.