Evidence for significant contribution of a newly identified monoamine transporter (PMAT) to serotonin uptake in the human brain
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter in the central nervous system (CNS) that influences a multitude of brain functions, including autonomic function, motor activity, hormone secretion, cognition, and complex processes associated with affection, emotion, and reward [1], [2]. Functional deficiency in 5-HT signaling has been implicated in the pathophysiology of various depressive syndromes [2], [3], [4]. A major goal of antidepressant therapy is to restore 5-HT signaling by elevating extracellular 5-HT levels in the brain [4], [5]. Consequently, genes involved in 5-HT clearance, such as the serotonin reuptake transporter (SERT) and the monoamine oxidases (MAOs), have long been considered as drug targets for the treatment of depression [5], [6]. For example, the widely used selective serotonin reuptake inhibitors (SSRIs) exert their pharmacological effects by specifically blocking SERT-mediated 5-HT reuptake.
Transporter-mediated cellular uptake plays a key role in determining the intensity and duration of 5-HT signaling [4], [7]. Earlier studies suggested that two different transport systems for 5-HT may exist, a high affinity-low capacity process and a low affinity-high capacity process, which are termed uptake1 and uptake2, respectively [8], [9], [10]. The uptake1 system for 5-HT is now known to be mediated by the Na+/Cl−-dependent, high affinity transporter, SERT. SERT is primarily expressed in serotonergic neurons and represents the major pathway for presynaptic 5-HT clearance [4], [7]. The low affinity uptake2 system is Na+-independent, displays broad substrate specificity towards monoamine neurotransmitters, and has not been well characterized [8], [11]. Recent cloning work suggests that the uptake2 system may consist of multiple transporters, including members of the organic cation transporters (OCT) family and the plasma membrane monoamine transporter (PMAT) recently identified in our laboratory [12], [13]. These non-SERT transporters may play a role in 5-HT clearance in specific brain regions and/or when extracellular concentrations of 5-HT reach high levels. Uptake2 transporters have also been proposed as potential drug targets for developing antidepression agents with improved efficacy [14].
The human PMAT cDNA encodes a protein of 530 amino acid residues with 11 putative transmembrane domains [12]. When expressed in mammalian cells or Xenopus laevis oocytes, PMAT exhibits typical uptake2 characteristics, mediating Na+-independent, low affinity and high capacity transport of monoamine neurotransmitters [12]. Among the monoamine neurotransmitters, PMAT has the highest affinity towards 5-HT (Km = 114 μM), as compared to dopamine, norepinephrine, and epinephrine (Km ranging from 330 to 15,000 μM) [12]. In cells stably expressing PMAT and SERT, the apparent affinity of recombinant PMAT for 5-HT is much lower than that of SERT (Km, 114 versus 0.5 μM) [12], [15]. However, PMAT also has a much larger transport capacity (Vmax), resulting in roughly comparable uptake efficiencies (Vmax/Km) to SERT in heterologous expression systems [12], [15]. Consistent with its transport function for monoamine neurotransmitters, PMAT mRNA is most strongly expressed in the human brain, and is widely distributed in the CNS [12]. Currently, the in vivo significance of PMAT in brain clearance of 5-HT in humans or intact animals is unknown as most of our previous studies were performed using cDNA transfected heterogenous express systems. In this study, we used an antisense hybrid depletion approach in X. laevis oocytes to investigate the functional significance of PMAT in the brain. This method has been widely used to evaluate the relative importance of a specific transporter to total tissue uptake [16], [17], [18], [19], [20]. Because our data suggested a significant role of PMAT in total brain 5-HT uptake, potential pharmacological interactions between the SSRIs and PMAT have also been investigated.
Section snippets
Chemicals
[3H]5-HT (specific radioactivity, 27.1 Ci/mmol) was from Perkin-Elmer Life Sciences Inc. (Boston, MA). [3H]MPP+ (specific radioactivity, 39.3 Ci/mmol) was from American Radiolabeled Chemicals Inc. (St. Louis, MO). Fluoxetine, paroxetine, and fluvoxamine were from Sigma (St. Louis, MO). Citalopram and sertraline were obtained from Cenrilliant Corporation (Round Rock, TX).
cRNA and mRNA expression in X. laevis oocytes
PMAT cDNA [12] was subcloned into an oocyte expression vector pOX under the control of T3 promoter using Hin dIII and Xba I
Functional expression of PMAT and SERT in oocytes and validation of oligos
PMAT cRNA was synthesized and injected into oocytes. Uptake was performed using [3H]5-HT (1 μM). Compared to water-injected oocytes, oocytes injected with PMAT cRNA exhibited ∼6-fold increase in 5-HT uptake (Fig. 2a). Pre-hybridization of PMAT cRNA with sense oligos (A–C) had no inhibitory effect on PMAT-mediated 5-HT uptake. In contrast, pre-hybridization of cRNA with antisense oligos (A–C) reduced 5-HT uptake levels to those observed in water-injected oocytes (Fig. 2a). These data suggest that
Discussions
In this study, we employed an antisense hybrid depletion approach in X. laevis oocytes to explore the functional significance of a newly cloned monoamine transporter PMAT in the human brain. Previously, the hybrid depletion approach has been used to estimate the relative contribution of a number of individual membrane transporters to tissue-specific uptake of a ligand [16], [17], [18], [19], [20]. This relatively simple and straightforward method allows for the evaluation of gene function in
Acknowledgements
This study was supported by a National Institutes of Health grant GM66233. MZ is supported in part by a predoctoral fellowship from Eli Lilly and Company Foundation. We would like to thank Dr. Randy Blakely at Vanderbilt University for providing the human SERT cDNA.
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Present address: Merck KGaA, Institute of Drug Metabolism and Pharmacokinetics, Am Feld 32, D-85567 Grafing, Germany.