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 site is located within TM1-6 and PMAT interaction with organic cations may involve aromatic residues. In this study, we analyzed the role of tyrosine and tryptophan residues located within TM1-6 with the goal to identify potential residues involved in substrate recognition and translocation. The six Tyr and one Trp residues in this region were each mutated to alanine followed by analysis of these mutants’ membrane localization and transport activity towards 1-methyl-4-phenylpyridinium (MPP+), serotonin (5-HT) and dopamine. Two mutants, Y85A and Y112A, showed normal cell surface expression but lost their transport activity towards organic cations. At position Y85, aromatic substitution with Phe or Trp fully restored organic cation transport activity. Interestingly, at position Y112, Phe substitution is not allowed. Trp substitution at Y112 partially restored transport activity towards 5-HT and dopamine, but severely impaired MPP+ transport. Detailed kinetic analysis revealed that Trp substitution at Y85 and Y112 affected apparent binding affinity (Km) and maximal transport velocity (Vmax) in a substrate-dependent manner. Together these 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 analysis also suggested the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.

Molecular Analysis and Structure-Activity Relationship Modeling of the Substrate/Inhibitor Interaction Site of Plasma Membrane Monoamine Transporter

“…These observations are consistent with our mutational analysis of PMAT, in which we identified a negatively charged residue (E206) in TM5 that functions as a charge sensor and is critical for cation selectivity (Zhou et al, 2007b). We also recently identified two aromatic residues, Y85 and Y112 in TM 1 and 2, respectively, as important molecular determinants for PMAT substrate interactions (Ho and Wang, 2010).…”

Interspecies comparison of the functional characteristics of plasma membrane monoamine transporter (PMAT) between human, rat and mouse

“…YFP was tagged to the flexible, long intracellular N-terminus of hPMAT or rPmat, and our previous studies have demonstrated that tagging of YFP to the N-terminus of hPMAT do not affect the substrate selectivity or kinetic behavior of hPMAT (Engel et al, 2004; Zhou et al, 2007b; Ho and Wang, 2010). Similarly, Okura et al also reported that N-terminal FLAG tagging did not interfere with the membrane localization or transport function of rPmat (Okura et al, 2011).…”

Residue Ile89 in human plasma membrane monoamine transporter influences its organic cation transport activity and sensitivity to inhibition by dilazep

“…Despite their distinct substrate profiles (i.e. organic cations versus nucleosides), previous studies suggest PMAT and ENTs share a similar protein structure with a common 11 TM membrane topology [16,17]. The major substrate recognition sites in PMAT and the ENTs are both located in the N-terminal half (TM1-6), and transplanting TM1-6 of PMAT into hENT1 converted hENT1 from a nucleoside transporter to an organic cation transporter [17].…”

“…The major substrate recognition sites in PMAT and the ENTs are both located in the N-terminal half (TM1-6), and transplanting TM1-6 of PMAT into hENT1 converted hENT1 from a nucleoside transporter to an organic cation transporter [17]. Recent mutational analyses further identified several residues (Y85, Y112, E206, T220) in TM1, 2 and 5 of PMAT critical for substrate recognition and translocation [16,17]. Interestingly, these residues are located at or close to the TM regions or residues that are known to be important for nucleoside transport in the ENTs [16,17].…”

“…Recent mutational analyses further identified several residues (Y85, Y112, E206, T220) in TM1, 2 and 5 of PMAT critical for substrate recognition and translocation [16,17]. Interestingly, these residues are located at or close to the TM regions or residues that are known to be important for nucleoside transport in the ENTs [16,17]. …”