Molecular Determinants of Substrate Selectivity of a Novel Organic Cation Transporter (PMAT) in the SLC29 Family
2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês
10.1074/jbc.m609421200
ISSN1083-351X
AutoresMingyan Zhou, Xia Li, Karen Engel, Joanne Wang,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoPlasma 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 Glu206 in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the charge at Glu206 (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. Thr220, another residue in TM5, also showed an effect on PMAT activity. Helical wheel analysis of TM5 revealed a distinct amphipathic pattern with Glu206 and Thr220 clustered in the center of the hydrophilic face. In summary, our results suggest that Glu206 functions as a critical charge sensor for cationic substrates and TM5 forms part of the substrate permeation pathway in PMAT. 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 Glu206 in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the charge at Glu206 (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. Thr220, another residue in TM5, also showed an effect on PMAT activity. Helical wheel analysis of TM5 revealed a distinct amphipathic pattern with Glu206 and Thr220 clustered in the center of the hydrophilic face. In summary, our results suggest that Glu206 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 (1Hediger M.A. Romero M.F. Peng J.B. Rolfs A. Takanaga H. Bruford E.A. Pflugers Arch. 2004; 447: 465-468Crossref PubMed Scopus (730) Google Scholar, 2Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 2000; 64: 354-411Crossref PubMed Scopus (683) Google Scholar). The solute carrier (SLC) 4The abbreviations used are: SLC, solute carrier; PMAT, plasma membrane monoamine transporter; ENT, equilibrative nucleoside transporter; OCT, organic cation transporter; YFP, yellow fluorescent protein; MPP+, 1-methyl-4-phenylpyridinium; MDCK, Madin-Darby canine kidney; WT, wild type. 4The abbreviations used are: SLC, solute carrier; PMAT, plasma membrane monoamine transporter; ENT, equilibrative nucleoside transporter; OCT, organic cation transporter; YFP, yellow fluorescent protein; MPP+, 1-methyl-4-phenylpyridinium; MDCK, Madin-Darby canine kidney; WT, wild type. proteins constitute a large series of membrane transporters currently consisting of 360 members in 46 gene families in humans (1Hediger M.A. Romero M.F. Peng J.B. Rolfs A. Takanaga H. Bruford E.A. Pflugers Arch. 2004; 447: 465-468Crossref PubMed Scopus (730) Google Scholar). 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 (1Hediger M.A. Romero M.F. Peng J.B. Rolfs A. Takanaga H. Bruford E.A. Pflugers Arch. 2004; 447: 465-468Crossref PubMed Scopus (730) Google Scholar, 2Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 2000; 64: 354-411Crossref PubMed Scopus (683) Google Scholar). 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 (2Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 2000; 64: 354-411Crossref PubMed Scopus (683) Google Scholar, 3Wright E.M. Loo D.D. Hirayama B.A. Turk E. Physiology (Bethesda). 2004; 19: 370-376Crossref PubMed Scopus (147) Google Scholar). For example, the Na+-glucose transporter family, SLC5, comprises not only Na+-coupled glucose transporters, but also transporters for iodide, choline, vitamins, and short-chain fatty acids (3Wright E.M. Loo D.D. Hirayama B.A. Turk E. Physiology (Bethesda). 2004; 19: 370-376Crossref PubMed Scopus (147) Google Scholar).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 (4Kong W. Engel K. Wang J. Curr. Drug Metab. 2004; 5: 63-84Crossref PubMed Scopus (221) Google Scholar, 5Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (590) Google Scholar). ENT1 and ENT2 play important roles in nucleoside salvage pathways, regulation of adenosine signaling, and cellular disposition of anticancer and antiviral nucleoside analogs (4Kong W. Engel K. Wang J. Curr. Drug Metab. 2004; 5: 63-84Crossref PubMed Scopus (221) Google Scholar, 5Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (590) Google Scholar). ENT3 is an intracellular nucleoside transporter, which may play a role in lysosomal transport of nucleosides (6Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). The fourth member, SLC29A4, was originally identified as ENT4 and was expected to function as a nucleoside transporter (7Acimovic Y. Coe I.R. Mol. Biol. Evol. 2002; 19: 2199-2210Crossref PubMed Scopus (75) Google Scholar). However, our recent cloning and characterization work clearly showed that SLC29A4 functions as a polyspecific organic cation transporter that transports prototype organic cations (e.g. 1-methyl-4-phenylpyridinium (MPP+), tetraethylammonium, and biogenic amines) but minimally interacts with nucleosides or nucleoside analogs (8Engel K. Wang J. Mol. Pharmacol. 2005; 68: 1397-1407Crossref PubMed Scopus (157) Google Scholar, 9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Because its typical endogenous substrates are monoamine neurotransmitters (e.g. dopamine, serotonin), we have proposed a functional name, plasma membrane monoamine transporter (PMAT), for SLC29A4 (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The substrate selectivity of PMAT is strikingly similar to that of the organic cation transporters (OCTs) in the SLC22 family (10Fujita T. Urban T.J. Leabman M.K. Fujita K. Giacomini K.M. J. Pharm. Sci. 2006; 95: 25-36Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 11Koepsell H. Endou H. Pflugers Arch. 2004; 447: 666-676Crossref PubMed Scopus (447) Google Scholar, 12Wright S.H. Dantzler W.H. Physiol. Rev. 2004; 84: 987-1049Crossref PubMed Scopus (347) Google Scholar, 13Zhang L. Brett C.M. Giacomini K.M. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 431-460Crossref PubMed Scopus (181) Google Scholar). In humans, PMAT may play a role in regulating central nervous system homeostasis of monoamine neurotransmitters (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 14Zhou M. Engel K. Wang J. Biochem. Pharmacol. 2007; 73: 147-154Crossref PubMed Scopus (57) Google Scholar), as well as in tissue-specific transport of organic cations (8Engel K. Wang J. Mol. Pharmacol. 2005; 68: 1397-1407Crossref PubMed Scopus (157) Google Scholar, 15Xia L. Engel K. Zhou M. Wang J. Am. J. Physiol. 2006; (in press)Google Scholar).The unique substrate selectivity of PMAT in the SLC29 family has brought up intriguing questions: why does PMAT behave so differently from the ENTs (i.e. ENT1–3) and is PMAT structurally related to the ENTs or to the OCTs? At the protein level, human and rodent PMATs exhibit a low overall sequence identity (∼20%) to the ENTs (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) (Table 1), which is on the borderline of classifying a transporter into a specific gene family. However, in the TM regions, sequence identity between PMAT and the ENTs significantly increases (up to 35–40%), and PMAT does not show significant sequence homology to other known gene families (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Its sequence identity to the OCTs is only 11–14% (Table 1). Moreover, PMAT and ENTs both possess an 11-transmembrane topology (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 16Sundaram M. Yao S.Y. Ingram J.C. Berry Z.A. Abidi F. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2001; 276: 45270-45275Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Finally, Barnes et al. (17Barnes K. Dobrzynski H. Foppolo S. Beal P.R. Ismat F. Scullion E.R. Sun L. Tellez J. Ritzel M.W. Claycomb W.C. Cass C.E. Young J.D. Billeter-Clark R. Boyett M.R. Baldwin S.A. Circ. Res. 2006; 99: 510-519Crossref PubMed Scopus (147) Google Scholar) recently showed that PMAT also transports adenosine at acidic pH. These data suggest an intrinsic relationship in protein structure between PMAT and the ENTs. In this study, we investigated the molecular determinants and structural elements underlying the unique substrate selectivity of PMAT in the SLC29 family.TABLE 1Sequence identity and similarity of PMAT with human ENTs and OCTs Open table in a new tab EXPERIMENTAL PROCEDURESSequence Alignment and Analysis—Sequences of mammalian ENTs and human OCTs were obtained from GenBank™ with the following accession numbers: hENT1 (AF_079117), rENT1 (NM_031684), mENT1 (AF_218255), hENT2 (AF_034102), rENT2 (NM_031738), mENT2 (AF_183397), hENT3 (AF_326987), mENT3 (AF_326986), PMAT (AY_485959), rPMAT (XP_001072979), mPMAT (NM_146257), hOCT1 (X_98332), rOCT1 (NP_036829), mOCT1 (NP_033228), hOCT2 (X_98333), rOCT2 (NP_113772), mOCT2 (NP_038695), hOCT3 (AJ_001417), rOCT3 (NP_062103), and mOCT3 (NP_035525). ClustalW and Vector NTI Suite 8 software were used for sequence analysis and multiple alignments.Generation of Chimeric and Mutant Constructs—To facilitate the determination of membrane localization, chimeras and mutants were constructed using yellow fluorescence protein (YFP)-tagged wild type (WT) PMAT and hENT1 as templates. The WT PMAT and hENT1 cDNAs (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) were subcloned into the YFP vector pEYFP-C1 (Clontech, Palo Alto, CA) using the BglII/XbaI and BglII/KpnI sites, respectively. To construct chimeras T1–6 and T7–11, an internal EcoRI site of hENT1 in the loop region between TM6 and 7 was used. An engineered EcoRI site was introduced into WT PMAT cDNA by site-directed mutagenesis, and chimeras were generated using a method described by Wang and Giacomini (18Wang J. Giacomini K.M. J. Biol. Chem. 1997; 272: 28845-28848Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To generate additional chimeras with smaller replacement, an engineered site was introduced into both WT human PMAT and hENT1. Junctions in all chimeras were made within homologous regions. No foreign amino acid was introduced to any of the chimera constructs. For site-directed mutagenesis, mutants were generated using the QuikChange kit (Stratagene) as described previously (19Wang J. Giacomini K.M. J. Biol. Chem. 1999; 274: 2298-2302Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The sequence of each chimera and mutant was confirmed by DNA sequencing in the Department of Biochemistry at the University of Washington.Stable Expression in MDCK Cells—YFP-tagged chimera and mutant constructs were transfected into MDCK cells using a Lipofectamine (Invitrogen) method as previously described (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 15Xia L. Engel K. Zhou M. Wang J. Am. J. Physiol. 2006; (in press)Google Scholar). Stably transfected cell lines were obtained by culturing cells in minimal essential medium containing 10% fetal bovine serum and G418 (1000 μg/ml). Empty pEYFP-C1 vector was transfected into MDCK cells to obtain the control cell line. After 2–3 weeks selection, fluorescence-positive cells were purified by a FACS Vantage SE sorter (BD Biosciences) at the Cell Analysis Center at the University of Washington Health Science Center. The sorted cells were cultured and maintained in minimal essential medium containing G418 (200 μg/ml).Confocal Fluorescence Microscopy—To determine the cellular localization of YFP-tagged chimeric and mutant transporters, 106 cells were grown in two-well Lab-Tek borosilicated coverglass chambers (Nalge Nunc International Corp.) and visualized with a Leica Spectral confocal microscope equipped with a krypton/argon laser as the light source. Images were captured by excitation at 488 nm and emission at 610–640 nm.Functional Characterization in MDCK Cells—Stably transfected MDCK cells were plated in 24-well plates and allowed to grow for 3 days. Transport assays were performed at 37 °C in Krebs-Ringer-Henseleit buffer (5.6 mm glucose, 125 mm NaCl, 4.8 mm KCl, 1.2 mm KH2PO4, 1.2 mm CaCl2, 1.2 mm MgSO4, 25 mm HEPES, pH 7.4) containing a 3H-labeled ligand. [3H]5-HT (5-hydroxy-[1,2-3H]tryptamine creatinine sulfate, 27.1 Ci/mmol) and [3H]dopamine (3,4-dihydroxy-[2,5,6-3H]phenylethylamine, 59.7 Ci/mmol) were from PerkinElmer Life Sciences, Inc. [3H]MPP+ (39.3 Ci/mmol) and [3H]uridine ([5,6-3H]uridine, 30.0 Ci/mmol) were from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Uptake was terminated by washing the cells three times with ice-cold Krebs-Ringer-Henseleit buffer. Cells were then solubilized and the radioactivity was quantified by liquid scintillation counting. Protein content in each uptake well was measured using a BCA protein assay kit (Pierce) and the uptake in each well was normalized to its protein content. In all studies, cells transfected with an empty vector were served as a background control. Transporter-specific uptake was calculated by subtracting the background uptake in vector-transfected cells.Isolation of Plasma Membrane Proteins by Cell Surface Biotinylation—Confluent cells were washed twice with 3 ml of ice-cold phosphate-buffered saline/CM (pH 8.0). Biotinylation was carried out on ice by incubation with 1 ml of ice-cold phosphate-buffered saline/CM containing a membrane-impermeable biotinylation reagent NHS-SS-biotin (0.5 mg/ml). After two successive 20-min incubations on ice with freshly prepared NHS-SS-biotin and gentle shaking, cells were briefly rinsed with 3 ml of phosphate-buffered saline/CM containing 100 mm glycine. Cells were then incubated on ice with the same solution for 20 min to ensure complete quenching of the unreacted NHS-SS-biotin. Cells were then solubilized on ice by incubating in 400 μl of lysis buffer (10 mm Tris, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Triton X-100, protease inhibitors phenylmethylsulfonyl fluoride, 200 μg/ml, leupeptin, 3 μg/ml, pH 7.4) for 1 h. Fifty microliters of streptavidin-agarose beads was then added to the supernatant to isolate cell membrane protein, which are subjected to Western blot using a mouse monoclonal anti-yellow fluorescent protein antibody (JL-8) (BD Biosciences) with 1:1000 dilution, followed by horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000 dilution).Helical Wheel Analysis—The helical wheel was generated using the Wisconsin Package version 10.3-UNIX as described before (19Wang J. Giacomini K.M. J. Biol. Chem. 1999; 274: 2298-2302Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The transmembrane domain is assumed to be a standard α-helix (3.6 residues/helical turn). Each residue in TM is plotted every 100° around the center of a circle. The projection of the positions of the residues was shown on a plane perpendicular to the helical axis. Hydrophobicity and hydrophilicity are assigned according to the consensus scale of Eisenberg et al. (20Eisenberg D. Wilcox W. McLachlan A.D. J. Cell. Biochem. 1986; 31: 11-17Crossref PubMed Scopus (62) Google Scholar).Data Analysis—All uptake experiments were performed in triplicate and repeated three times. Data were expressed as mean ± S.D. Statistical significance was determined by Student's t test, and the kinetic parameters were determined by nonlinear least squares regression fitting as described previously (8Engel K. Wang J. Mol. Pharmacol. 2005; 68: 1397-1407Crossref PubMed Scopus (157) Google Scholar).RESULTSFunction and Substrate Selectivity of PMAT and hENT1 Chimeric Transporters—Among all known human genes, PMAT only shows a low but significant sequence homology to ENTs (9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) (Table 1). Multiple computer algorithms also predicted that PMAT possesses a similar membrane topology to the ENTs with 11 putative transmembrane helices, a cytoplasmic N terminus, and an extracellular C terminus (Fig. 1a). Based on these data, we hypothesized that despite the difference in substrate selectivity, PMAT and ENTs may share a similar tertiary structure and a similar arrangement of functional domains for substrate recognition and translocation. Earlier studies have suggested that the N-terminal half (TM1–6) of ENT1 and ENT2 contains critical domains for substrate/inhibitor recognition and translocation (21Sundaram M. Yao S.Y. Ng A.M. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1998; 273: 21519-21525Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 22Yao S.Y. Ng A.M. Vickers M.F. Sundaram M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2002; 277: 24938-24948Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To test our hypothesis, we first constructed two chimeric transporters, chimeras T1–6 and T7–11, between human PMAT and the prototype human equilibrative nucleoside transporter, hENT1 (Fig. 1). Chimera T1–6 consists of the N-terminal half (TM1–6) of PMAT and the C-terminal half (TM7–11) of hENT1, whereas chimera T7–11 was constructed in a reciprocal manner (Fig. 1a). To monitor membrane trafficking of chimeric proteins, YFP was tagged to the N termini of both chimeric and WT transporters. After being stably transfected into MDCK cells, the substrate specificity of the chimeras were determined by uptake assays using MPP+ and uridine for the PMAT and hENT1 phenotype, respectively. YFP-tagged WT PMAT and hENT1 were expressed predominantly on the plasma membranes in MDCK cells. Tagging of YFP had little effect on substrate selectivity and kinetic behaviors of WT transporters (data not shown) (23Lai Y. Bakken A.H. Unadkat J.D. J. Biol. Chem. 2002; 277: 37711-37717Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). As expected, cells expressing WT PMAT showed uptake activity for MPP+ but not for uridine (Fig. 1b). In contrast, cells expressing WT hENT1 transported uridine but not MPP+. Chimera T1–6 showed plasma membrane localization (data not shown) and behaved like PMAT, transporting MPP+ but not uridine (Fig. 1b). The PMAT-like substrate selectivity of chimera T1–6 strongly suggests that TM1–6 contains the key domains for substrate recognition and translocation in PMAT. The reciprocal chimera T7–11, however, was not functional due to intracellular trapping in MDCK cells as revealed by confocal microscopy (data not shown). Further attempts to make chimeras with smaller replacements were not successful, as all resultant chimeras were not functional when expressed in MDCK cells (data not shown).Selection of Candidate Amino Acid Residues for Site-directed Mutagenesis—To further identify regions and residues important for the substrate selectivity of PMAT, we employed sitedirected mutagenesis based on multiple sequence alignments. Because PMAT has been shown to transport a variety of organic cations (8Engel K. Wang J. Mol. Pharmacol. 2005; 68: 1397-1407Crossref PubMed Scopus (157) Google Scholar, 9Engel K. Zhou M. Wang J. J. Biol. Chem. 2004; 279: 50042-50049Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), we proposed that negatively charged residues are important for its cation selectivity, particularly those conserved in PMAT orthologs but substituted by a noncharged or positively charged residues in the ENTs. Of the 33 glutamate (Glu) and aspartate (Asp) residues conserved in human and rodent PMATs, 13 residues were not taken into consideration because they are located in the very hydrophilic N or C terminus or the large intracellular loop linking TM6 and -7. Among the remaining 20 negatively charged residues, 10 showed charge differences from mammalian ENTs and were thus selected for mutagenesis. Fig. 2a shows an example of two selected residues in TM3 and TM5. In addition, we also performed forced sequence alignments of PMATs with human and rodent OCT1–3. Despite the low sequence homology between PMATs and the OCTs, there are 23 conserved residues. Among these residues, Thr226 in the OCTs was previously suggested to participate in the binding of organic cations (24Popp C. Gorboulev V. Muller T.D. Gorbunov D. Shatskaya N. Koepsell H. Mol. Pharmacol. 2005; 67: 1600-1611Crossref PubMed Scopus (121) Google Scholar). We thus included the equivalent residue in PMAT, Thr220, for mutational analysis. Together, 11 residues, Asp91, Asp107, Glu128, Asp154, Asp163, Glu206, Thr220, Glu227, Glu242, Asp336, and Glu375 were chosen as candidate residues. As shown in Fig. 2b, 9 of the 11 residues are located in the N-terminal half (TM1–6) and only two are located in the C-terminal half in TM7.FIGURE 2a, examples of multiple sequence alignment of PMATs (ENT4s) with mammalian ENTs in TM3 and TM5 regions. The residues Asp154 and Glu206 are conserved in PMATs (red) but are substituted with uncharged residues in the ENTs (yellow). b, positions of candidate residues selected for alanine substitution.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of Candidate Residues by Alanine Substitution— Alanine substitution was used to test the functional significance of the 11 candidate residues. YFP-tagged PMAT mutants (D91A, D107A, E128A, D154A, D163A, E206A, T220A, E227A, E242A, D336A, and E375A) were constructed and stably expressed in MDCK cells. The function of these alanine mutants was analyzed by uptake studies using PMAT substrates MPP+, serotonin, and dopamine. As shown in Fig. 3, mutation of Asp91 and Glu128 to alanine did not result in a significant change in transport activity. Mutants E227A and E375A were also functional and had a slightly increased (∼1.5–2-fold) transport activity. Mutants E242A and T220A exhibited reduced transport activity that was about 30% of the WT PMAT. In contrast, mutants D107A, D154A, D163A, E206A, and D336A completely lost transport activity toward all three tested substrates.FIGURE 3Uptake of organic cations by various PMAT alanine mutants in stably transfected MDCK cells. The uptake of 10 μm 3H-labeled MPP+, dopamine, and serotonin was measured at 37 °C for 1 min. Substrate uptake was corrected by subtracting nonspecific uptake in vector-transfected cells. Values are expressed as percentage of uptake in cells expressing wild type YFP-PMAT measured in parallel. Each value represents the mean ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cellular Localization of Alanine Mutants—The observed change in the uptake activity of an alanine mutant could be due to a change in intrinsic transport activity or an alteration of cell surface expression levels of the functional transporter. To distinguish these two circumstances, confocal microscopy was used to visualize the cellular localization of all tested alanine mutants (Fig. 4). For mutants D91A, E128A, T220A, E227A, E242A, and E375A, which retained full or partial transport activity, a predominant plasma membrane localization pattern was observed (Fig. 4). Mutants D107A, D154A, D163A, and D336A, which are not functional in MDCK cells, displayed altered cellular localization. D154A and D336A were localized intracellularly to perinuclear membrane network, indicating that substitutions with alanine affected PMAT trafficking to the plasma membrane. The other two mutants, D107A and D163A, showed diffused fluorescence throughout the cytoplasm, which was similar to the distribution of YFP. Western blot analysis revealed degraded protein products in cells expressing D107A and D163A (data not shown), suggesting that the alanine substitutions at these two positions may have affected protein stability. Most importantly, mutant E206A, which showed complete loss of transporter activity (Fig. 3), exhibited normal plasma membrane expression (Fig. 4). These data suggest that unlike D107A, D154A, D163A, and D336A, the loss of function in the E206A mutant is not caused by protein trafficking or stability problems, but rather is due to a defect in transport activity.FIGURE 4Confocal imaging of cellular localization of wild type PMAT, YFP, and various PMAT alanine mutants in stably transfected MDCK cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Role of Glu206 in the Interactions with MPP+ and Monoamine Neurotransmitters—To further investigate the role of Glu206 in PMAT interactions with cationic substrates, three additional mutants, E206Q, E206D, and E206R, were constructed and stably expressed in MDCK cells. All mutants exhibited plasma membrane expression patterns similar to that of the WT PMAT (Fig. 5a). Cell surface biotinylation followed by Western analysis with anti-yellow fluorescent protein antibody also revealed comparable membrane expression of the 75-kDa YFP-tagged fusion protein in all Glu206 mutant cell lines (Fig. 5b). Functional analysis showed that elimination of the negative charge (E206Q) or switching to a positively charged residue (E206R) resulted in complete loss of transporter activity (Fig. 6). In contrast, substitution of Glu206 with a charge-conserved residue (E206D) generated a fully functional transporter (Fig. 6) with similar transport efficiency toward various cationic substrates (Table 2). These data strongly suggest that the negative charge at position 206 is essential for the transport function of PMAT, and organic cation binding to PMAT may involve electrostatic interaction with Glu206.FIGURE 5Plasma membrane expression of Glu206 and Thr220 mutants as revealed by confocal fluorescence microscopy (a) or biotinylation followed by Western blot with an anti-yellow fluorescent protein monoclonal antibody (b).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Uptake of organic cations by Glu206 and Thr220 mutants in stably transfected MDCK cells. The uptake of 10 μm 3H-labeled MPP+, dopamine, and serotonin was measured at 37 °C for 1 min. Substrate uptake was corrected by subtracting nonspecific uptake in vector-transfected cells. Values are ex
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