Precise control of monoamine neurotransmitter levels in the extracellular fluids of the brain is critical in maintaining efficient and robust neurotransmission. High affinity transporters in the solute carrier SLC6A family function in removing monoamines from the neurosynaptic cleft. Emerging evidence suggests that these transporters are only one part of a system of transporters that work in concert to maintain brain homeostasis of monoamines. Here we report the cloning and characterization of a new human plasma membrane monoamine transporter, PMAT. The PMAT cDNA encodes a protein of 530 amino acid residues with 10 -12 transmembrane segments. PMAT is not homologous to known neurotransmitter transporters but exhibits low homology to members of the equilibrative nucleoside transporter family. When expressed in Madin-Darby canine kidney cells and Xenopus laevis oocytes, PMAT efficiently transports serotonin (K m ‫؍‬ 114 M), dopamine (K m ‫؍‬ 329 M), and the neurotoxin 1-methyl-4-phenylpyridinium (K m ‫؍‬ 33 M). In contrast, there is no significant interaction of PMAT with nucleosides or nucleobases. PMAT-mediated monoamine transport does not require Na ؉ or Cl ؊ but appears to be sensitive to changes in membrane potential. Northern blot analysis showed that PMAT is predominantly expressed in the human brain and widely distributed in the central nervous system. These studies demonstrate that PMAT may be a novel low affinity transporter for biogenic amines, which, under certain conditions, might supplement the role of the high affinity transporters in the brain.In the central nervous system, monoamine neurotransmitters, including dopamine, serotonin, and norepinephrine, control a variety of physiological, behavioral, and endocrine functions (1, 2). Monoamine-mediated neurotransmission is also critically involved in a number of brain pathological processes such as Parkinson's disease, depression, schizophrenia, and drug addiction (1, 2). A key step that determines the intensity and duration of monoamine signaling is the transport of released monoamines into brain cells. This process is carried out by cell surface transporters that transport monoamines into presynaptic nerve terminals or neighboring glial cells where they can be recycled through repackaging into secretory vesicles or degraded by intracellular enzymes (3-5).Uptake of released monoamines into presynaptic neurons is mainly carried out by a family of Na ϩ -and Cl Ϫ -dependent high affinity plasma membrane transporters, which includes the dopamine transporter (DAT), 1 the serotonin transporter (SERT), and the norepinephrine transporter (NET) (3-5). These transporters, which share high sequence similarity and belong to the solute carrier 6A (SLC6A) family, are the known targets for many psychostimulants, antidepressants, and neurotoxins (3-5).Several lines of evidence suggest that in addition to the SLC6A high affinity transporters (i.e. DAT, SERT, and NET), the brain expresses other transporters to regulate extracellular monoamine levels. First, a number of...

Many endogenous compounds and xenobiotics are organic cations that rely on polyspecific organic cation transporters (OCTs) to traverse cell membranes. We recently cloned a novel human plasma membrane monoamine transporter (PMAT) that belongs to the equilibrative nucleoside transporter (ENT) family. We have reported previously that, unlike other ENTs, PMAT (also known as ENT4) is a Na ϩ -independent and membrane potential-sensitive transporter that transports monoamine neurotransmitters and the neurotoxin 1-methyl-4-phenylpyridinium (MPP ϩ ). These compounds are the known substrates for OCTs, which raises the possibility that PMAT functions as a polyspecific transporter like the OCTs. In the present study, we analyzed the interaction of PMAT with a series of structurally diverse organic cations using MDCK cells stably expressing human PMAT. Our study showed that PMAT interacts with many organic cations that have heterogeneous chemical structures. PMAT transports classic OCT substrates, such as tetraethylammonium, guanidine, and histamine. Prototype OCT inhibitors, including cimetidine, and type II cations (e.g., quinidine, quinine, verapamil, and rhodamine123) are also PMAT inhibitors. An analysis of molecular structures and apparent binding affinities revealed that charge and hydrophobicity are the principal determinants for transporter-substrate/ inhibitor interaction. A planar aromatic mass seems to be important for high affinity interaction. trans-Stimulation and efflux studies demonstrate that PMAT is able to mediate bidirectional transport. These functional properties of PMAT are strikingly similar to those of the OCTs. We therefore conclude that PMAT can function as a polyspecific organic cation transporter, which may play a role in organic cation transport in vivo.

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.Membrane transporters play pivotal roles in sustaining the normal life of cells (1, 2). The solute carrier (SLC) 4 proteins constitute a large series of membrane transporters currently consisting of 360 members in 46 gene families in humans (1). 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 (1, 2). 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 (2, 3). For example, the Na ϩ -glucose transporter family, SLC5, comprises not only Na ϩ -coupled glucose transporters, but also transporters for iodide, choline, vitamins, and short-chain fatty acids (3).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 (4, 5). ENT1 and ENT2 play important roles in nucleoside salvage pathways, regulation of adenosine signaling, and cellular disposition of a...

. Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells.

Nucleoside transporters mediate cellular uptake of physiologic nucleosides for nucleic acid synthesis in the salvage pathways in many cell types. These transporters also play an important role in in vivo disposition and intracellular targeting of many nucleoside analogs used in anticancer and antiviral drug therapy. In mammalian cells, there are two major nucleoside transporter gene families: the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The ENTs are facilitated carrier proteins and the CNTs are Na(+)-dependent secondary active transporters. Recent molecular cloning of a number of ENT and CNT transporters has greatly advanced our understanding of the molecular and cellular mechanisms by which nucleosides and nucleoside analogs are transported across biological membranes. In this manuscript, we review the structure, function, tissue distribution, and cellular localization of various cloned mammalian nucleoside transporters. Information on transporter interaction with various nucleoside drugs and analogs is presented. Current knowledge on the regulation of nucleoside transporters in various cell types and tissues is reviewed. The therapeutic significance of nucleoside transporters is discussed along with emerging data from recent clinical studies.