Tyrosine 112 is essential for organic cation transport by the plasma membrane monoamine transporter

Abstract

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter in the solute carrier 29 (SLC29) family. Previous studies suggested that the major substrate recognition sites are located within transmembrane domains (TM) 1-6, and interaction of PMAT with organic cations may involve aromatic residues. In this study, we analyzed the roles of tyrosine and tryptophan residues located within TM1-6 with a goal of identifying potential residues involved in substrate recognition and translocation. The six tyrosines and one tryptophan in this region were each mutated to alanine followed by analysis of the mutant's membrane localization and transport activity toward 1-methyl-4-phenylpyridinium (MPP(+)), serotonin (5-HT), and dopamine. Two mutants, Y85A and Y112A, exhibited normal cell surface expressions but lost their transport activities toward organic cations. At position Y85, aromatic substitution with phenylalanine or tryptophan fully restored organic cation transport activity. Interestingly, at position Y112, phenylalanine substitution was not allowed. Tryptophan substitution at Y112 partially restored transport activity toward 5-HT and dopamine but severely impaired MPP(+) transport. Detailed kinetic analyses revealed that tryptophan substitution at Y85 and Y112 affected the apparent binding affinity (K(m)) and maximal transport velocity (V(max)) in a substrate-dependent manner. Together, our data suggest that Y85 and Y112 are important molecular determinants for PMAT function, and Y112 is indispensable for optimal interaction with organic cation substrates. Our analyses also suggest the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.

Figure 1

Proposed secondary structure of PMAT. Positions of candidate aromatic residues selected for mutagenesis analysis are highlighted in red.

Figure 2

(a) Uptake of ³H-labeled MPP⁺ (1 μM), 5-HT (10 μM) and dopamine (10 μM) by WT PMAT and various alanine mutants in stably transfected MDCK cells. Uptake was performed at 37°C for 4 min. Substrate uptake was corrected by subtracting nonspecific uptake in vector-transfected cells. Values are expressed as percentage of uptake in cells expressing WT PMAT. Each value represents the mean ± S.D. from three independent experiments (n=3) with different cell passages. For each experiment, uptake was carried out in triplicates in three different wells on the same plate. (b) Confocal imaging of cellular localization of WT PMAT, pEYFP-C1, and various PMAT alanine mutants in stably transfected MDCK cells. (c) Plasma membrane expression of WT PMAT and various PMAT alanine mutants detected by biotinylation followed by Western blot with an anti-yellow fluorescent protein monoclonal antibody.

Figure 3

(a) Confocal imaging of cellular localization of WT PMAT and various Y85 mutants in stably transfected MDCK cells. (b) Upper panel, uptake of ³H-labeled MPP⁺ (1 μM), 5-HT (10 μM) and dopamine (10 μM) by WT PMAT and various Y85 mutants in stably transfected MDCK cells. Uptake was performed at 37°C for 4 min. Substrate uptake was corrected by subtracting nonspecific uptake in vector-transfected cells. Values are expressed as percentage of uptake in cells expressing WT PMAT. Each value represents the mean ± S.D. from three independent experiments (n=3) with different cell passages. For each experiment, uptake was carried out in triplicates in three different wells on the same plate. *, p<0.01, vs WT values. Lower panel, plasma membrane expression of WT PMAT and various Y85 mutants detected by biotinylation followed by Western blot with an anti-yellow fluorescent protein monoclonal antibody.

Figure 4

(a) Confocal imaging of cellular localization of WT PMAT and various Y112 mutants in stably transfected MDCK cells. (b) Upper panel, uptake of ³H-labeled MPP⁺ (1 μM), 5-HT (10 μM) and dopamine (10 μM) by WT PMAT and various Y112 mutants in stably transfected MDCK cells. Uptake was performed at 37°C for 4 min. Substrate uptake was corrected by subtracting nonspecific uptake in vector-transfected cells. Values are expressed as percentage of uptake in cells expressing WT PMAT. Each value represents the mean ± S.D. from three independent experiments (n=3) with different cell passages. For each experiment, uptake was carried out in triplicates in three different wells on the same plate. *, p<0.01, vs WT values. #, p<0.01, vs Y112W-mediated 5-HT and dopamine uptake values. Lower panel, plasma membrane expression of WT PMAT and various Y112 mutants detected by biotinylation followed by Western blot with an anti-yellow fluorescent protein monoclonal antibody.

Figure 5

Concentration-dependent transport of (a) MPP⁺, (b) 5-HT, and (c) dopamine by WT PMAT, Y85W and Y112W. Vector-, WT PMAT-, Y85W-, and Y112W-transfected MDCK cells were incubated with varying concentrations of the substrates for 1 min at 37°C. Specific uptake was calculated by subtracting the uptake values in vector-transfected cells. WT PMAT (●), Y85W (□), and Y112W (Δ) concentration-dependent uptake were shown. Each value represents the mean ± S.D. from three independent experiments (n=3) with different cell passages. For each experiment, uptake was carried out in triplicates in three different wells on the same plate.

Figure 6

(a) Multiple sequence alignment of PMATs with mammalian ENTs in TM1 and TM2 regions. Y85 and Y112 in PMATs and their corresponding residues on ENTs are numbered and highlighted in yellow. Functionally important residues previously identified in ENTs are numbered and highlighted in red. Corresponding residues in human PMAT are numbered. (r: rat; m: mouse; ca: canine; c: cattle; mac: macaques; h: human; rb: rabbit) (b) Uptake of uridine in the presence of NBMPR by various PMAT mutants in stably transfected MDCK cells. Values are expressed as pmol/mg protein/min. Each value represents the mean ± S.D. from three independent experiments (n=3) with different cell passages. For each experiment, uptake was carried out in triplicates in three different wells on the same plate. There was no statistically difference in uridine transport for WT and mutant PMAT proteins as compared to the pEYFP-C1 vector-transfected cells.

Figure 7

Helical wheel analysis of (a) TM1 and (b) TM2 of PMAT. The transmembrane domains are assumed to be standard α-helix and each residue is plotted every 100° around the center of a circle. The figures show the projection of the positions of the residues on a plane perpendicular to the helical axis. Hydrophobic residues are shown in white, and hydrophilic residues are shown in gray. Residues in PMAT identified to be functionally important in this study are marked by asterisks*. Functionally important residues previously identified in ENT1 and/or ENT2 with positions corresponding or close to Y85 and Y112 in PMAT are indicated in bracket.