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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.

SUBMITTER: Ho HT

PROVIDER: S-EPMC2995015 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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

Ho Horace T B HT Wang Joanne J

Biochemistry 20100901 36

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 recognitio ...[more]

PMID: 20687515

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Project description:Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane α-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl]methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH 8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.

Dataset Information

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

Publications

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

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