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.
Highlights
Constitute a large series of membrane transporters currently consisting of 360 members in 46 gene families in humans [1]
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-methyl4-phenylpyridinium (MPPϩ), tetraethylammonium, and biogenic amines) but minimally interacts with nucleosides or nucleoside analogs [8, 9]
The unique substrate selectivity of plasma membrane monoamine transporter (PMAT) in the SLC29 family has brought up intriguing questions: why does PMAT behave so differently from the equilibrative nucleoside transporter (ENT) (i.e. ENT1–3) and is PMAT structurally related to the ENTs or to the organic cation transporters (OCTs)? At the protein level, human and rodent PMATs exhibit a low overall sequence identity (ϳ20%) to the ENTs [9] (Table 1), which is on the borderline of classifying a transporter into a specific gene family
Summary
Sequence Alignment and Analysis—Sequences of mammalian ENTs and human OCTs were obtained from GenBankTM 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). 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. 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 [18]. Stable Expression in MDCK Cells—YFP-tagged chimera and mutant constructs were transfected into MDCK cells using a Lipofectamine (Invitrogen) method as previously described [9, 15]. 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). 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. Statistical significance was determined by Student’s t test, and the kinetic parameters were determined by nonlinear least squares regression fitting as described previously [8]
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