Molecular Determinants of Substrate Selectivity of a Novel Organic Cation Transporter (PMAT) in the SLC29 Family

Abstract

Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1-3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1-6 of PMAT and TM7-11 of hENT1 behaved like PMAT, transporting 1-methyl-4-phenylpyridinium (MPP ؉ , an organic cation) but not uridine (a nucleoside), suggesting that TM1-6 contains critical domains responsible for substrate recognition. To identify residues important for the cation selectivity of PMAT, 10 negatively charged residues were chosen and substituted with alanine. Five of the alanine mutants retained PMAT activity, and four were non-functional due to impaired targeting to the plasma membrane. However, alanine substitution at Glu 206 in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the charge at Glu 206 (E206Q) resulted in loss of organic cation transport activity, whereas conserving the negative charge (E206D) restored transporter function. Interestingly, mutant E206Q, which possesses the equivalent residue in ENT1, gained uridine transport activity. Thr 220 , another residue in TM5, also showed an effect on PMAT activity. Helical wheel analysis of TM5 revealed a distinct amphipathic pattern with Glu 206 and Thr 220 clustered in the center of the hydrophilic face. In summary, our results suggest that Glu 206 functions as a critical charge sensor for cationic substrates and TM5 forms part of the substrate permeation pathway in PMAT.Membrane transporters play pivotal roles in sustaining the normal life of cells (1, 2). The solute carrier (SLC) 4 proteins constitute a large series of membrane transporters currently consisting of 360 members in 46 gene families in humans (1). Transporters from the same SLC gene family share at least 20 -25% amino acid sequence identity and are thought to be evolved from a common ancestor (1, 2). Whereas it is generally anticipated that transporters from the same gene family possess similar functional properties, several SLC families appear to consist of members with great functional diversity (2, 3). For example, the Na ϩ -glucose transporter family, SLC5, comprises not only Na ϩ -coupled glucose transporters, but also transporters for iodide, choline, vitamins, and short-chain fatty acids (3).We recently discovered an interesting functional divergence in the equilibrative nucleoside transporter (ENT) family, SLC29. The human and rodent genomes encode four SLC29 isoforms, SLC29A1-4. SLC29A1-3, known as ENT1-3, are nucleoside transporters that specifically transport nucleosides (e.g. uridine, adenosine, etc.) and their structural analogs (4, 5). ENT1 and ENT2 play important roles in nucleoside salvage pathways, regulation of adenosine signaling, and cellular disposition of a...

Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleoside nephrotoxicity

“…We previously showed that PMAT functions as a Na ϩ -independent polyspecific transporter whose substrate specificity is remarkably similar to the OCTs (4). While PMAT does not typically interact with other nucleosides and nucleoside analogs, adenosine is recognized and transported by PMAT, probably in the protonated form (1,30). In the present study, we determined the intrarenal cellular localization of PMAT using a newly developed peptide antibody.…”

“…Plasma membrane protein extract from vector-or PMAT-transfected MDCK cells was prepared and subjected to Western blot analysis as previously described (30). To verify that P469 does not react with other SLC29 isoforms, Western blot was carried out in cells overexpressing yellow fluorescence protein-tagged ENT1 (SLC29A1) using MDCK cell lines previously generated in this laboratory (30). To detect PMAT expression in human kidney, commercial blots containing human kidney whole lysates (20 g/lane) were purchased from Imgenex (San Diego, CA).…”

Nucleoside transporters in PET imaging of proliferating cancer cells using 3ꞌ-deoxy-3ꞌ-[18F]fluoro-L-thymidine

“…hENTs are widely distributed in most, possibly all, cell types and hENTs 1-3 have broad specificity for both purine and pyrimidine nucleosides. hENT4, also known as PMAT, is uniquely selective for adenosine and also transports a variety of organic cations [16][17][18][19][20]. Some nucleosidederived drugs can also interact with and be translocated by members of the SLC22 gene family, which include organic anion transporters (OATs), organic cation transporters (OCTs) and organic carnitine and zwitterion transporters (OCTNs) [9,[21][22][23][24][25].…”

Ligand-gated Ion Channels

“…ENT1-3 are described as broad-spectrum nucleoside transporters. Ahas been reported to be intracellular purine nucleoside transporters Endogenous substrates Adenosine, guanosine, inosine, uridine, thymidine, cytidine, hypoxanthine, adenine, thymine (Yao et al, 2011) Adenosine, guanosine, inosine, uridine, thymidine, hypoxanthine Adenosine, inosine, > guanosine, thymidine, uridine, adenine (Baldwin et al, 2005) 5HT, dopamine > tyramine, histamine (Engel and Wang, 2005) Synthetic substrates PMAT also transports adenosine at acidic pH (Barnes et al, 2006;Zhou et al, 2007).…”