Biogenic amine transporters: regulation in flux

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Biogenic amine transporters: regulation in flux Randy D Blakely* and Andrea L Bauman Following vesicular release, the biogenic amine neurotransmitters dopamine, norepinephrine and serotonin are actively cleared from extracellular spaces by presynaptic transporters. These transporters interact with multiple psychoactive agents including cocaine, amphetamines and antidepressants. Recent findings indicate that amine reuptake is likely to be a tightly regulated component of synaptic plasticity rather than a constitutive determinant of transmitter clearance. Protein kinase C activation and transporter phosphorylation have been linked to regulatory protein trafficking, and both phosphorylation and trafficking may be influenced by transporter ligands. Recognition that transmitters, antagonists and second messengers can modify the intrinsic activity, surface expression or protein levels of amine transporters raises new questions about the fundamental nature of drug actions in vivo. The theory that dysregulation of transporters may contribute to disease states is supported by the recent discovery that a coding mutation in the human norepinephrine transporter contributes to orthostatic intolerance. Addresses Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232–6420, USA *e-mail: [email protected] Current Opinion in Neurobiology 2000, 10:328–336 0959-4388/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations 5-HT ADHD BGT DA DAT DLG EL GABA GAT NE NET NSF PDZ domain PKC PP PROT PSD-95 SERT SNARE SNP SSRI TMD ZO-1

5-hydroxytryptamine, serotonin attention deficit/hyperactivity disorder betaine transporter dopamine DA transporter discs large extracellular loop γ-aminobutyric acid GABA transporter norepinephrine NE transporter N-ethylmaleimide-sensitive factor PSD-95, DLG, ZO-1 domain protein kinase C protein phosphate proline transporter postsynaptic density protein of 95 kDa serotonin transporter soluble NSF attachment protein receptor single nucleotide polymorphism selective serotonin reuptake inhibitor transmembrane domain zona occludens 1

Introduction The biogenic amine neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) are very simple molecules with highly complex actions in the peripheral and central nervous systems ranging

from the control of heart rate to the coloring of mood. Pharmacologists have been fascinated by the amines for decades, as the management of amine production, action or inactivation figures prominently in the treatment of autonomic, emotional and cognitive disturbances. The past decade began with an elucidation of the genes responsible for clearance of amines from the synaptic cleft [1]. Evaluation of the amino acid sequence of cloned NE transporters (NETs) [2] led to the prediction that transporters comprised 12 transmembrane domains (TMDs), in register with the hydrophobic sequences of the GAT1 GABA transporter [3]. This model, subsequently adopted for cloned DA transporters (DATs) and 5-HT transporters (SERTs), identified a large hydrophilic extracellular loop (EL2) between TMD3 and 4 and placed the NH2 and COOH termini intracellularly. The initial cloning of amine transporters also revealed multiple protein phosphorylation sites on the cytoplasmic domains. With a growing awareness of the sensitivity of amine transport to kinase activation and the generation of heterologous expression systems as well as transporter-specific antibodies suitable for biochemical and cell biologic studies, amine transporter biology has entered a new era of discovery. Recent studies, which will be reviewed here, indicate that regulation of transporter function and surface expression can be rapidly modulated and that drugs previously studied only for their impact on transport activity may have unrecognized capacities for influencing transporter regulation. The impact that such regulatory processes have on human physiology and behavior may be revealed by recent studies identifying the first coding mutation in an amine transporter that supports a disease process.

Regulating amine reuptake: general thoughts Pharmacological studies with amine transporter knockout mice reveal a requirement for presynaptic amine transporter expression for normal transmitter clearance, presynaptic transmitter homeostasis and drug responses [4,5••,6••]. These studies also indicate that cells must make both quantitative (how much, how active) and qualitative (which transporter to express, where to locate them) decisions to achieve appropriate transporter contributions for the particular neurotransmitter signals they modulate. The influence that transporters have over biogenic amine signaling depends on three major factors: the kinetics of receptor signal transduction mechanisms and their propensity for desensitization; the density and localization of transporters (and receptors) expressed at the plasma membrane; and the intrinsic activity of individual transporters. If receptor activation and desensitization were to occur on a millisecond time scale, transporter binding of substrates, rather than transport per se, would be more likely to influence postsynaptic responses. Biogenic amine neurotransmitters, however, (with the exception of synapses employing 5-HT3 ligand-gated channels) signal through G-protein-coupled

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receptors with relatively slow kinetics. Signaling to effectors is thus slower than membrane potential changes observed for small molecule neurotransmitters like glutamate, GABA, glycine and acetylcholine, which act via ligand-gated channels. Over a period of a few minutes of constant stimulation, many G-protein-coupled receptors desensitize, placing both the time course of effector influences and receptor regulation on a time frame compatible with the proposed turnover rates of amine transporters (1–10/sec). Thus, it seems likely that both the abundance as well as the intrinsic turnover rates of biogenic amine transporters could be modulated with physiologic consequences, with the regulation of the transporters depending on the particular spatial organization of reuptake and release sites and the density and desensitization kinetics of receptors. Finally, when other properties of transporters are also considered (ion channel activity, efflux; see below), we find that qualitative changes in exactly what the transporters do while they are at the membrane may also be subject to regulation.

Transporter trafficking: skimming the surface of transporter regulation Prior to the 1990s, methods for investigating the distribution and surface expression of biogenic amine transporters were unavailable, perhaps perpetuating the concept that surface expression is set at a constitutive level and is not subject to modulation. In the mid 1990s, with the arrival of transporterspecific antibodies and the development of heterologous expression models, researchers began to report evidence for changes in transport capacity that ensue rapidly (1–30 min) following the activation of cellular kinases. The most common observation was that application of protein kinase C (PKC) activators leads to a reduction in amine transport capacity, revealed by a change in transport capacity (Vmax) with little or no change in substrate dependence (Km) [7]. These changes in transport capacity following PKC activation appear to be specific, as other membrane proteins and transport activities measured concurrently exhibit little, no, or opposite changes over the same time course.

Transporter surface expression Recent studies with transporter-specific antibodies and/or epitope-tagged constructs have yielded evidence that changes in transport capacity arise from changes in transporter surface distribution. Initial evidence for altered trafficking as a route for amine transporter modulation has come from studies of SERT proteins in stably transfected HEK-293 cells [8] and homologous GAT1 GABA transporters expressed in Xenopus oocytes [9,10]. Apparsundaram and coworkers have visualized a PKCdependent redistribution of NET proteins in transfected LLC-PK1 cells [11]; in additon, a redistribution of surface DAT proteins following PKC activation has been reported in transfected COS [12•] and PC12 [13••] cells and cRNAinjected Xenopus oocytes [14]. Recently, green fluorescent protein (GFP)-tagged DATs expressed in MDCK cells have enabled the real-time visualization of DAT internalization following PKC activation [15••]. Other pathways, including

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those under the influence of cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase (PKG), Ca2+/calmodulin-dependent pathways and tyrosine kinases have also been reported [7,16], suggesting that amine and related transporters are under the control of a variety of signal transduction pathways that dictate the final level of surface transporter expression.

Transporter endocytosis: recycling or degradation? What is the fate of amine transporters once they leave the plasma membrane? Melikian and Buckley [13••], using transfected PC-12 cells, report a colocalization of internalized DAT proteins with transferrin receptors, proteins known to actively recycle to the plasma membrane. These findings suggest the passage of DATs through a recycling endosomal compartment. In contrast, Daniels and Amara [15••] report the movement of internalized DATs to lysosomes for degradation. It is possible that observations of NET degradation following chronic treatments of cells in vitro with NET-specific antidepressants relate to these findings [17•]. Similarly, Benmansour and coworkers [18•] provide evidence of a loss of SERT binding sites in rats exposed to chronic selective serotonin reuptake inhibitor (SSRI) administration (paroxetine or sertraline) that is not a result of altered SERT gene expression. Studies examining SERT protein levels are underway to explore whether these findings result from a residual drug effect or from alterations in transporter protein levels. What accounts for the different itineraries for DAT proteins following PKC activation (recycling endosomes versus lysosomes) [15••]? The use of protease inhibitors by Daniels and Amara [15••] to eliminate contributions from de novo synthesis may have inadvertently biased trafficking to a lysosomal itinerary — the normal fate may have been for the DATs to recycle more often to the plasma membrane. Conceivably, Daniels and Amara have gained evidence for a labile protein or protein complex that normally shunts internalized transporters back to the cell surface after endocytosis. Alternatively, these conflicting data could relate to the different contexts of DAT expression (pheochromocytoma versus polarized epithelial cell) in the two studies. Polarized epithelial cells may recycle proteins from apical membranes (DAT is apically expressed) differently from proteins in basolateral membranes and, as such, the DAT itinerary may be a consequence of its initial targeting in this heterologous model system. Thus, one might expect that DATs could behave quite differently in other expression systems (and in neurons). Perhaps this is also why Zhang and coworkers [19•] found that chronic cocaine treatment modulates DAT antagonist binding and transport activity in transfected neuroblastomas but not in transfected COS cells. Future studies are warranted to settle these issues and to determine how the cell context influences the trafficking of amine transporters in response to PKC activation and transporter ligands. Nonetheless, these studies establish regulated cell surface trafficking as an acute response to kinase activation, particularly the activation of PKC.

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Regulation of endogenous transporters The studies reviewed above use heterologous expression in transfected cell lines or oocytes assayed in vitro. Although more limited in number, studies with isolated tissue preparations reveal similar acute transporter regulatory events that not only can be triggered by activated protein kinases but also by direct auto- and heteroreceptor activation. Apparsundaram and coworkers report muscarinic M3ACh-receptor-triggered internalization of NET proteins in SK-N-SH cells, partially mediated by a PKClinked pathway [20••]. Endogenous DAT activity appears to be regulated by presynaptic D2 receptors. Importantly, D2-receptor knockout mice reveal a loss of DAT modulation by D2 antagonists [21••]. These and earlier in vitro studies suggest that D2 receptor activation enhances DAT expression. Perhaps presynaptic D2 receptors reduce release of DA and increase its uptake as part of a coordinated regulatory network to influence synaptic DA availability. The signaling pathway that supports this modulation is currently unknown. 5-HT transport capacity in platelets and brain tissue appears to be under the influence of histamine and adenosine receptors, respectively (see [7] for a recent review). Daws and colleagues [22••], using in vivo amperometric techniques, have provided evidence of 5-HT1b receptormodulated SERT activity in rat hippocampus. Like D2 modulation of DATs, 5HT1b receptor activation appears to stabilize or enhance 5HT clearance from extracellular spaces, an effect that is detected when these receptors are blocked with antagonists. As with D2 modulation of DATs, whether 5HT1b modulation of SERT activity is supported by altered transporter trafficking is unknown. In addition, the signal transduction pathways leading from receptor to transporter are not yet understood. In studies of related proteins, Quick and colleagues [23•] have provided robust evidence that multiple PKC-linked receptors are capable of modulating the functional capacity of GAT1 GABA transporters in cultured neurons; the effect of this modulation is a redistribution of GAT1 protein from the cell surface to intracellular membranes. From these studies we conclude that the regulation of neurotransmitter transporters evoked by direct kinase activators and second messengers is likely to mirror the regulation that occurs as a result of receptor activation. Further studies are warranted to clarify which receptors regulate which transporters, whether transporters are regulated through dedicated signaling pathways, and whether transporter regulation is always tightly linked to changes in the release process.

Transporter phosphorylation: the signal for transporter internalization? Many signaling proteins, including receptors and ion channels, are modulated via direct protein phosphorylation. Over the past several years, evidence has accumulated to support a role of biogenic amine transporter phosphorylation in transporter regulation. Huff, Vaughan and

coworkers first demonstrated that DATs expressed in LLC-PK1 cells [24] and in synaptosomes [25] can be phosphorylated following PKC activation and protein phosphatase inhibition. Recently, Ramamoorthy and coworkers demonstrated that phosphorylated SERT proteins are immunoprecipitated from transfected HEK-293 cells following activation of PKC, PKA or PKG and also following selective protein phosphatase (PP1/2A) inhibition [26••]. Interestingly, SERT phosphorylation ensuing from PP1/2A inhibition in HEK-293 cells is not blocked by PKC, PKA or PKG antagonists, suggesting that an, as yet, unidentified kinase is responsible for SERT phosphorylation. NET phosphorylation is also evident in immunoprecipitates from transfected LLC-PK1 cells following PKC activation (S Apparsundaram, unpublished data). Although phosphorylation of biogenic amine transporters is evident under conditions that lead to transporter redistribution, a requirement for phosphorylation has not been demonstrated as neither the stoichiometry of the reaction nor the sites of phosphorylation have been defined.

Substrate-modulated transporter phosphorylation It is possible that the key kinase target responsible for transporter redistribution is not the transporter per se but an associated protein or protein complex. Recent studies of substrate modulation of SERT phosphorylation and trafficking by Ramamoorthy and Blakely [27••], strengthen the case for direct transporter phosphorylation as the key to transporter trafficking. These authors report that 5-HT and amphetamine substrates block the ability of SERTs to become phosphorylated following PKC activation. Moreover, 5-HT coincubation with PKC activators limits transporter internalization. Dampened phosphorylation is not a result of cytoplasmic 5-HT accumulation but appears to correlate with substrate occupancy and translocation. The activity-dependence of transporter regulation provides strong evidence that PKC-dependent SERT phosphorylation occurs at the plasma membrane; thus, phosphorylation is compartmentalized in a manner which is appropriate for its use as a signal for transporter internalization. Whether 5-HT influences the exposure of phosphorylation sites or the activities of transporter-associated kinases and phosphatases is unknown but is an active area of inquiry. The fact that amphetamines prevent SERT phosphorylation whereas antagonists permit it may also shed new light on the actions of these drugs following chronic exposure in vivo. Whereas Ramamoorthy and Blakely report effects of 5HT on SERT phosphorylation and trafficking, Daniels and Amara, using MDCK cells, fail to observe DA-dependent modulation of DAT trafficking [15••]. These latter studies suggest either that DATs utilize substrates differently from SERTs for regulation of surface trafficking or that the ability to respond to substrates is dependent on the cell context of expression. Studies of DAT phosphorylation in

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this model have not been presented and it remains possible that a phosphorylation-independent regulatory process controls DAT expression in this system. In studies analogous to those investigating the activity-dependent modulation of SERTs, Bernstein and Quick [28••] report that GAT1 GABA transporters are also subject to usedependent modulation of transport capacity and surface expression in hippocampal neuronal cultures.

Transporter-associated proteins Phosphorylation-dependent desensitization and trafficking of G-protein-coupled receptors involves dynamic physical associations of receptors with kinases, phosphatases and adaptor proteins. We and others have proposed analogous schemes for the regulated trafficking of biogenic amine transporters [7,29]. Recent findings from our group (A Bauman, unpublished data) reveal associations between SERT, NET and DAT proteins and the catalytic subunit (c) of PP2A (PP2Ac). SERT–PP2Ac associations can be regulated by PKC activation, suggesting that transporter phosphorylation may arise not only from activated kinases but also from the dissociation of protective protein phosphatases. Integral membrane proteins also appear to form complexes with transporters. GAT1 GABA transporters physically associate in native and heterologous systems with the target (t)-SNARE syntaxin-1 in a PKC-dependent manner [30••]. By use of toxins (e.g. botulinum C) that cleave syntaxin selectively, as well as syntaxin/GAT1 cotransfection experiments, Beckman and coworkers report that interactions with syntaxin-1 are essential for GAT1 modulation by PKC. Members of our laboratory have gathered similar evidence for a PKCdependent association of NET proteins with syntaxin-1 in heterologous models and noradrenergic tissues (U Sung, S Apparsundaram, unpublished data), suggesting that these findings may generalize to the biogenic amine transporters. In our studies, phorbol esters trigger a dissociation of the syntaxin-1–NET complex. Whether this regulation arises via activated PKC or via one of a number of new phorbol ester targets in cells (e.g. UNC-13) is unknown. Studies are also required for the determination of the generality of syntaxin associations with other biogenic amine transporters and, most importantly, for elucidating whether transporter phosphorylation drives syntaxin disassembly or whether transporter phosphorylation is dependent on prior complex dissociation.

Transporters as homomultimeric complexes The models discussed above relate to heteromeric assemblies of transporters with other membrane or cytosolic proteins. Recent evidence indicates, however, that homomultimeric assemblies of transporter proteins may be at the core of the transporter complex, and such assemblies must be considered as we attempt to visualise the coassembly of transporter regulators. Chang and coworkers [31•] exploited heterologously expressed, concatamer cDNA constructs to lend functional support to the idea of multimeric SERTs. The recent work of

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Kilic and Rudnick [32••] has added weight to these studies; the authors have identified SERT multimeric protein complexes by immunoprecipitation of differentially tagged cDNAs. Multimeric assemblies are evident in biotinylated membrane fractions that are indicative of surface expression. Moreover, functional interactions are evident in coexpression experiments of mutant subunits that either bear or lack sites for inactivation by membraneimpermeant reagents [32••]. Clearly, these findings are critical for our attempts to model the physical basis of transporter regulation. Whether each monomer is functionally independent for the different behaviors we now associate with biogenic amine transporters (neurotransmitter movement, leak currents, substrate-gated current, pH-modulated current, etc.) is unknown. It is also unclear how assembly occurs and whether endoplasmic reticulum retention signals, noted in other multimeric channel proteins [33], are critical in retaining unassembled monomers. Reports of transporter multimerism in transfected systems also suggest the possibility of complex functional interactions between splice variant cDNAs and polymorphic variants in vivo. Kitayama reported that endogenous splice variants of rat NET proteins exhibit dominant-negative behavior in heterologous co-expression studies [34•]. Perhaps these data reflect an endogenous mode of transporter regulation. Whether this occurs for human NET splice variants [35•] is unknown. Splice variants of DAT or SERT proteins have yet to be identified, though with the possibility of multimer formation, it is likely that a more deliberate search will ensue.

Trafficking-independent regulation of biogenic amine transporters Cells could silence or activate surface transporters, rather than shuttling active carriers from one compartment to another, to achieve an equivalent functional result. Ion channels and G-protein-coupled receptors are subject to trafficking-independent modes of regulation, and it would be surprising if transporters did not also avail themselves of this opportunity. Detection of such regulation requires that one be able to demonstrate either altered function without a change in surface expression or a fundamental alteration in the properties of transport that cannot be explained by changes in the number of surface transporters. As noted, there are only a few reports of changes in substrate dependence (Km) and these changes tend to coincide with alterations in transport capacity (Vmax). Perhaps we are simply not measuring the relevant parameters for shifts in transporter function. For example, amine transporters are known to support a significant nonstoichiometric ion flow that is gated by the transmitter. We have been struck by how complete and rapid the loss of 5-HT-gated currents can be following phorbol ester treatments of SERT-transfected HEK-293 cells, as compared with the slower, less complete losses of transport capacity and transporter surface expression [8]. Whether changes in transporter currents reflect transporter phosphorylation or altered

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protein–protein associations (such as through Ca2+/calmodulin interactions with transporter proteins or syntaxins) is presently unclear. Jayanthi and coworkers note a similar dissociation of current modulation versus uptake regulation in studies of Ca2+-modulated proline transport [36•]. In these studies, changes in intracellular Ca2+ evoke a rapid elevation in proline transporter (PROT) currents within seconds of Ca2+ elevation, an effect that has been linked through inhibitor studies to Ca2+/calmodulin-dependent protein kinase II (CaM-KII) activation. Changes in transport, in contrast, are detected only after many minutes of Ca2+ elevation and the effect is inhibitory, not stimulatory. Modulated transporters could shift between states where substrate-gated currents are permitted and ones where they are not, providing additional routes for the alteration of cell physiology. The remarkable finding that arachidonic acid induces large cocaine-sensitive currents in DAT proteins [37•] suggests this idea may not be far-fetched. Gnegy’s group may also have uncovered evidence for altered function independent of transporter trafficking in studies of native DAT proteins following application or injection of PKC antagonists [38••]. Acute treatment of striatal slices with multiple PKC antagonists leads to a loss of amphetamine-evoked DA release. Similar effects are seen with in vivo injection of the PKC antagonist Ro31-8220 [39•]. If these effects arise solely from a change in DAT surface expression, one would expect to see commensurate changes in DAT-mediated uptake, which are not observed. Thus, PKC-dependent phosphorylation of DAT could shift the activity of DATs from supporting inward transport exclusively to an enhanced tendency for amphetamine-evoked efflux. If this phosphorylation is maintained, transporters may then be internalized and dephosphorylated and then returned to the cell surface or, perhaps, degraded. Clearly, further study of the molecular determinants of efflux is warranted in concert with phosphorylation and trafficking studies. The finding that efflux and influx for DATs may be differentially regulated, like transport and currents, suggests that there may be multiple mechanisms to regulate transporter function, only some of which are supported by altered protein trafficking.

Transporter polarity: putting carriers in their place Microscopic examination of DAT, NET and SERT localization in native tissues reveals targeting to axonal membranes and selected dendritic compartments but not to cell soma membranes [40,41•,42•]. This subcellular localization or polarized membrane expression presumably indicates a need to place transporters proximal to release sites. For NETs, Schroeter and coworkers report a punctate localization along many noradrenergic fibers [42•], though other fibers exhibit more continuous immunofluorescent labeling. SERT proteins have been found to be highly expressed on extrasynaptic axonal membranes [41•] and exhibit a remarkable polarity along the membranes of rat adrenal chromaffin cells [43]. In the

latter study, only membranes of chromaffin cells apposed to neighboring chromaffin cells exhibit SERT labeling, suggesting that cytoskeletal specializations that arise as a consequence of homophilic cell contacts may help target or stabilize SERTs in the adrenal gland. In polarized MDCK cells, SERT and NET proteins are enriched at the basolateral membrane, whereas DAT proteins localize to the apical membrane. These examples of polarized transporter expression — whether in neurons, neuroendocrine cells or epithelial cells — is evidence of organized associations between transporters and cytoskeletal proteins. Recently, Perego and coworkers [44 ••] have provided support for this hypothesis in studies of the basolateral localization of the betaine transporter (BGT-1) in MDCK cells. Although BGT-1 carriers are delivered to both apical and basolateral membranes, BGT-1 carriers, which are in the same gene family as DATs, NETs and SERTs, appear to be retained preferentially at the basolateral membrane through associations with PDZ domain proteins. The carboxyl terminus of BGT-1 was found to interact with the PDZ domain protein LIN-7 (Lineage-7 gene product), which polarizes to the basolateral membrane independently of BGT-1. Interestingly, the distal carboxyl termini of DAT, NET and SERT proteins are conserved across species variants. The carboxyl terminus of the Drosophila SERT protein bears a type I PDZ binding motif (Thr/Ser-X-Val, where X is any amino acid) and the mammalian SERTs have diverged only slightly (Asn-Ala-Val). We have found that the carboxyterminal acid of NET proteins is critical for appropriate levels of NET expression in a cell context-dependent manner (P Bauman, unpublished data), suggesting that proteins analogous or homologous to LIN-7 may play a role in the targeting or stability of amine transporters at membrane subdomains. Such protein complexes may also influence or mediate the ability of receptor stimulation to influence transporter expression.

Transporter genes as candidates for autonomic, emotional and cognitive disorders The recent progress in the cloning of human transporter genes and the observed changes in amine levels, receptors, behavior and drug responses of transporter-deficient mice have given researchers new impetus to look for genetic variants in transporter proteins that underlie behavioral or autonomic disorders. Promoter variants have been uncovered in the human SERT gene [45] that have an impact on mRNA and protein expression in vitro and in vivo. These variants have been linked to anxiety traits [45], autism [46,47], affective disorders [48] and alcoholism [49•], though not all studies reach similar conclusions — as might be expected from the complex nature of the behaviors being studied. Polymorphisms in the DAT gene of unknown functional significance have been linked to attention deficit/hyperactivity disorder (ADHD) [50,51•], which is intriguing given that DATdeficient mice exhibit the characteristically paradoxical calming response to methylphenidate (Ritalin) that is

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seen with ADHD patients [52••]. To date, no coding variants of DAT or SERT proteins have been linked with behavioral or physiologic traits. Recently, a NET coding variant has been identified in a patient with orthostatic intolerance (OI) [53••]. The affected subject is heterozygous for a TMD9 mutation (A457P) that abrogates function of NET in transfected cells. The phenotypic hallmarks of OI — tachycardia on standing, elevated plasma NE and decreased DHPG production — are evident in family members carrying the A457P allele. These studies describe the first disease known to arise through a biogenic amine transporter coding alteration. Additional NET, DAT, SERT and GAT1 mutations have recently been described through efforts to identify informative single nucleotide polymorphisms (SNPs) in the human genome [54•,55•]. These SNPs, as well as others, are now under careful evaluation for their impact on transporter biosynthesis, trafficking and regulation. As with ion channelopathies, we can anticipate that transporter polymorphisms will be discovered that not only abolish function, but which also increase transporter expression, influence voltage or ion-dependence, or alter the important regulatory sites that are required for normal transporter modulation.

Conclusions The past few years have been an active time for researchers evaluating the structure, function and regulation of transporter genes and proteins. Though much work remains to be done, we no longer consider transport as a constitutive property of synaptic membranes but rather as an actively regulated element of aminergic signaling. Transporters appear to be susceptible to regulatory influences mediated by protein associations and activated signal transduction cascades, and we are beginning to recognize that drugs we traditionally depict as modulators of amine reuptake also may have unanticipated actions on transporter regulation. Perhaps further study of transporter regulation will reveal new opportunities for pharmacologic manipulation of amine clearance in the form of modulated transporter phosphorylation and trafficking. Finally, as new genomic tools for high-throughput analysis of transporter genes arrive, additional transporter polymorphisms that result in aminergic dysfunction are likely to emerge.

Update Following the completion of this review, Xu and coworkers [56••] have reported the first findings with NET knockout mice. These mice, as expected, have a diminished clearance capacity for NE and exhibit behaviors seen with antidepressant-treated mice. Moreover, compensatory changes in dopaminergic signaling are evident including locomotor hyperresponsiveness to psychostimulants (cocaine and amphetamine) and DA receptor supersensitivity.

Acknowledgements The authors acknowledge the support of National Institutes of Health awards DA 07390 and MH 58291 (RD Blakely).

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References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Povlock SL, Amara SG: The structure and function of norepinephrine, dopamine, and serotonin transporters. In Neurotransmitter Transporters: Structure, Function, and Regulation. Edited by Reith MEA. Humana Press: Totowa NJ; 1997:1-28.

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Pacholczyk T, Blakely RD, Amara SG: Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 1991, 350:350-354.

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Guastella J, Nelson N, Nelson H, Czyzyk L, Keynan S, Miedel MC, Davidson N, Lester HA, Kanner BI: Cloning and expression of a rat brain GABA transporter. Science 1990, 249:1303-1306.

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Giros B, Jaber M, Jones SR, Wightman RM, Caron MG: Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 1996, 379:606-612.

5. ••

Jones SR, Gainetdinov RR, Jaber M, Giros B, Wightman RM, Caron MG: Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc Natl Acad Sci USA 1998, 95:4029-4034. DAT-deficient mice are found to exhibit significant alterations in DA levels, DA synthesis and DA turnover, revealing that appropriate levels of DAT expression dicates other presynaptic determinants of DA signaling. 6. ••

Bengel D, Murphy DL, Andrews AM, Wichems CH, Feltner D, Heils A, Mossner R, Westphal H, Lesch KP: Altered brain serotonin homeostasis and locomotor insensitivity to 3,4methylenedioxymetamphetamine (‘ecstasy’) in serotonin transporter-deficient mice. Mol Pharmacol 1998, 53:649-655. This is the first description of the phenotypes associated with genetically established SERT-deficiency. As with DAT knockouts, SERT disruption influences presynaptic neurotransmitter levels and biosynthesis of 5-HT. SERTdeficient animals fail to display MDMA-induced locomotor activation, supporting the proposal that SERTs are a target for the psychostimulant. 7.

Blakely RD, Ramamoorthy S, Schroeter S, Qian Y, Apparsundaram S, Galli A, DeFelice LJ: Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins. Biol Psych 1998, 44:169-178.

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Qian Y, Galli A, Ramamoorthy S, Risso S, DeFelice LJ, Blakely RD: Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J Neurosci 1997, 17:45-47.

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Corey JL, Davidson N, Lester HA, Brecha N, Quick MW: Protein kinase C modulates the activity of a cloned γ-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter. J Biol Chem 1994, 269:14759-14767.

10. Quick MW, Corey JL, Davidson N, Lester HA: Second messengers, trafficking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1. J Neurosci 1997, 17:2967-2979. 11. Apparsundaram S, Schroeter S, Blakely RD: Acute regulation of norepinephrine transport. II. PKC-modulated surface expression of human norepinephrine transporter proteins. J Pharmacol Exp Ther 1998, 287:744-751. 12. Pristupa ZB, McConkey F, Liu F, Man HY, Lee FJ, Wang YT, Niznik HB: • Protein kinase-mediated bidirectional trafficking and functional regulation of the human dopamine transporter. Synapse 1998, 30:79-87. The authors use confocal microscopy to show that a PKC-mediated decrease in DA uptake in human hDAT-transfected COS cells is a result of transporter internalization. In addition, the increase in transport activity seen with PKC or PKA inhibition is shown to result from the recuritment of intracellular transporters to the surface. These findings reveal bidirectional alterations in the transporter trafficking that supports DAT regulation. 13. Melikian H, Buckley K: Membrane trafficking regulates the activity of •• the human dopamine transporter. J Neurosci 1999, 19:7699-7710. Cell fractionation and biotinylation techniques are used to establish the pathway for steady-state as well as PKC-mediated DAT endocytosis in transfected PC-12 cells. DAT is shown to be trafficked along with transferrin receptors through recycling endosomes, suggesting that PKC activation enhances internalization of DA transporters through a recycling pathway that is constitutively active in PC-12 cells.

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14. Zhu SJ, Kavanaugh MP, Sonders MS, Amara SG, Zahniser NR: Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in Xenopus oocytes. J Pharmacol Exp Ther 1997, 282:1358-1365. 15. Daniels GM, Amara SG: Regulated trafficking of the human •• dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters. J Biol Chem 1999, 274:35794-35801. The authors provide an analysis of phorbol ester-regulated DAT trafficking in live cells using a green fluorescent protein (GFP)-tagged DAT construct. PMA (phorbol ester)-induced, clathrin-mediated endocytosis of DAT relocates DATs to early endosomal compartments. Using protein-synthesis inhibition to diminish the synthesis and insertion of new transporters, Daniels and Amara show that DATs are degraded after internalization in this paradigm; this degradation is mediated by a lysosomal pathway. 16. Hoffman BJ, Hansson SR, Mezey E, Palkovits M: Localization and dynamic regulation of biogenic amine transporters in the mammalian central nervous system. Front Neuroendocrinol 1998, 19:187-231. 17. •

Zhu M-Y, Blakely RD, Apparsundaram S, Ordway GA: Downregulation of the human norepinephrine transporter in intact 293-hNET cells exposed to desipramine. J Neurochem 1998, 70:1547-1555. Continuous and chronic exposure of cells to an NET-selective antidepressant (desipramine) in vitro leads to a loss of NE-transport capacity. Studies with NET-specific antibodies indicate a selective loss of NET protein. It is possible that NET conformations that target the protein for internalization and degradation are stabilized by the antidepressant (see [15••]). 18. Benmansour S, Cecchi M, Morilak DA, Gerhardt GA, Javors MA, • Gould GG, Frazer A: Effects of chronic antidepressant treatments on serotonin transporter function, density, and mRNA level. J Neurosci 1999 19:10494-10501. This study provides evidence for the in vivo downregulation of SERT function following chronic SSRI treatments. Effects on SERTs are selective for the particular SSRI used. The fact that no change in transporter mRNA or serotonin levels was seen suggests that loss of antagonist binding sites may reflect an alteration in SERT protein abundance or trafficking. 19 •

Zhang L, Elmer LW, Little KY: Expression and regulation of the human dopamine transporter in a neuronal cell line. Brain Res Mol Brain Res 1998, 59:66-73. Treatment of DAT-transfected Neuro-2A neuroblastomas with 1 µM cocaine for 24 h is reported to result in increased DAT-antagonist binding and DA uptake — these findings are not replicated in DAT-transfected COS-7 cells in the same study. These studies support an influence of ligand occupancy on biogenic amine transporter trafficking, but suggest that the ultimate effects on trafficking are dependent on the context of DAT expression, perhaps through the presence of different DAT-interacting proteins. 20. Apparsundaram S, Galli A, DeFelice LJ, Hartzell HC, Blakely RD: •• Acute regulation of norepinephrine transport: I. PKC-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells. J Pharmacol Exp Ther 1998, 287:733-743. This study provides evidence for G-protein-coupled receptor-mediated regulation of catecholamine transport in a noradrenergic neuroblastoma. Intracellular calcium- and PKC-linked pathways are implicated in M3 receptor actions on NETs. Changes in surface antagonist binding are consistent with receptor-modulated surface expression of NET proteins. 21. Dickinson SD, Sabeti J, Larson GA, Giardina K, Rubinstein M, •• Kelly MA, Grandy DK, Low MJ, Gerhardt GA, Zahniser NR: Dopamine D2 receptor-deficient mice exhibit decreased dopamine transporter function but no changes in dopamine release in dorsal striatum. J Neurochem 1999, 72:148-156. Evidence is provided for D2-receptor-mediated control of constitutive and regulated DAT expression. Analysis of D2 deficient mice reveals a loss of D2-receptor modulation of DATs in vivo. This study is the first to use receptor-deficient mice to demonstrate a role for a specific G-protein coupled receptor in transporter regulation. 22. Daws LC, Gerhardt GA, Frazer AL: 5-HT1b antagonists modulate •• clearance of extracellular serotonin in rat hippocampus. Neurosci Lett 1999, 266:165-168. In vivo chronoamperometry techniques are used to show that 5-HT1b antagonists decrease the clearance rate for 5-HT in the CA3 region of rat hippocampus. This provides evidence that serotonin autoreceptors can regulate the activity of SERTs in vivo. 23 •

Beckman ML, Bernstein EM, Quick MW: Multiple G protein-coupled receptors initiate protein kinase C redistribution of GABA transporters in hippocampal neurons. J Neurosci 1999, 19:RC9 (1-6). Activation of three different G-protein coupled receptors signaling through PKC activation is shown to reduce GABA uptake in cultured

hippocamal neurons. Both kinetic analysis of transport and surfacebiotinylation experiments show that the decrease in uptake corresponds to a shift in surface distribution of GAT1 from the plasma membrane to intracellular fractions. These findings reveal that GAT1 proteins, like SERT, NET and DAT proteins, are regulated through classical G-proteincoupled receptor signaling pathways. 24. Huff RA, Vaughan RA, Kuhar MJ, Uhl GR: Phorbol esters increase dopamine transporter phosphorylation and decrease transport Vmax. J Neurochem 1997, 68:225-232. 25. Vaughan RA, Huff RA, Uhl GR, Kuhar MJ: Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes. J Biol Chem 1997, 272:15541-15546. 26. Ramamoorthy S, Giovanetti E, Qian Y, Blakely RD: Phosphorylation •• and regulation of antidepressant-sensitive serotonin transporters. J Biol Chem 1998, 273:2458-2466. The authors of this study use immunoprecipitation analyses in human hSERT stably-transfected HEK-293 cells to provide the first evidence for SERT phosphorylation by multiple kinases. The findings support the idea that multiple signaling pathways linked to the activation of protein kinases (PKC, PKA, PKG) and protein phosphatases (PP2A) support regulation of SERT expression. These findings and those of Huff and Vaughan [24,25], support a role for phosphorylation in the acute regulation of biogenic amine transporter expression. 27. ••

Ramamoorthy S, Blakely RD: Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants. Science 1999, 285:763-766. The authors demonstrate activity-dependent modulation of SERT phosphorylation and surface expression in stably transfected HEK-293 cells. The substrates 5-HT and amphetamine — but not antagonists such as antidepressants and cocaine — were able to block PKC-mediated increases in transporter phosphorylation as well as transporter sequestration. The authors propose a pathway by which ligands can sustain or prevent PKCdependent transporter modulation either through conformational stabilization of SERTs or through an influence on SERT-associated proteins. 28. Bernstein EM, Quick MW: Regulation of γ-aminobutyric acid •• (GABA) transporters by extracellular GABA. J Biol Chem 1999, 274:889-895. Both GABA and GAT1 antagonists are shown in cultured hippocampal neurons to influence the surface distribution of GAT1 protein and thereby influence GABA transport capacity. Antagonists produce a decrease in transporter expression, whereas treatment with substrates results in a rapid increase in transporter expression. This study, like that of Ramamoorthy and Blakely involving SERTs [27••], proposes a mechanism whereby extracellular neurotransmitter can influence clearance capacity in a receptor-independent manner. 29. Beckman ML, Quick MW: Neurotransmitter transporters: regulators of function and functional regulation. J Membr Biol 1998, 164:1-10. 30. Beckman ML, Bernstein EM, Quick MW: Protein kinase C regulates •• the interaction between a GABA transporter and syntaxin 1A. J Neurosci 1998, 18:6103-6112. This study identifies a regulatable association between the SNARE protein syntaxin 1A and the GABA transporter GAT1. Using endogenous and transfected models, they show that SNARE–transporter interactions are influenced by PKC activation, suggesting that phorbol ester-induced transporter downregulation is a consequence of the availability of syntaxin for interacting with GAT-1. 31. Chang AS, Starnes DM, Chang SM: Possible existence of • quaternary structure in the high-affinity serotonin transport complex. Biochem Biophys Res Commun 1998, 249:416-421. Initial observations that truncation mutants of mouse mSERT coexpressed with wild-type exhibit a dominant-negative effect led this group to investigate the possibility of SERT oligomerization. Concatenates of mSERT cDNA reveal that productive transport is possible when dimeric and tetrameric, but not trimeric, constructs are formed (although trimeric constructs still bind cocaine analogs); this suggests that the protein may function as a dimer or tetramer. Though indirect, the studies lend support to the idea that a higher-order homomultimeric complex supports 5-HT transport. 32. Kilic F, Rudnick G: Oligomerization of the serotonin transporter •• and its functional consequences. Proc Natl Acad Sci USA 2000, in press. Co-immunoprecipitation studies of differentially tagged SERT proteins reveal direct evidence for multimer formation. Multimers are recovered from cell-surface fractions, as identified by cell-surface biotinylation. A dominant activity of subunits with accessible cysteines for methanethiosulfonate (MTS) inactivation is described; this is consistent with a tetrameric structure for SERT proteins.

Biogenic amine transporters Blakely and Bauman

33. Zerangue N, Schwappach B, Jan YN, Jan LY: A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 1999, 22:537-548. 34. Kitayama S, Ikeda T, Mitsuhata C, Sato T, Morita K, Dohi T: Dominant • negative isoform of rat norepinephrine transporter produced by alternative RNA splicing. J Biol Chem 1999, 274:10731-10736. Two isoforms of rat rNET are cloned from rat brain; these isoforms represent alternatively spliced gene products. rNETa corresponds to the original human hNET isolate and is a functional 12 TMD protein. rNETb contains a longer carboxyl terminus and has no transport activity in transfected cells. rNETb acts in a dominant-negative fashion when coexpressed with rNETa. Although a limit to the specificity of this dominant-negative effect was defined, as rNETa significantly reduces the activity of other members of the gene family, rNETa does not influence expression of glutamate transporters. These findings support the possibility of NET multimer formation and suggest that coexpression of splice variant cDNAs in vivo may be associated with complex functional properties. 35. Porzgen P, Bonisch H, Hammermann R, Bruss M: The human • noradrenaline transporter gene contains multiple polyadenylation sites and two alternatively spliced C-terminal exons. Biochim Biophys Acta 1998, 1398:365-370. A carboxy-terminal splice variant of unknown function is identified in SK-N-SH cells. Exons supporting differential carboxy-terminal splicing are identified. These studies support the idea that the carboxyl terminus of the human NET protein may be alternatively spliced although with a somewhat different pattern than that observed in bovine and rat NETs. 36. Jayanthi LD, Wilson JJ, Montcalvo J, DeFelice LJ: Differential • regulation of mammalian brain-specific proline transporter by calcium and calcium-dependent protein kinases. Brit J Pharmacol 2000, 129:465-470. This study reports differential influences of thapsigargin-triggered Ca2+ elevation on proline transport versus proline-transporter (PROT) dependent currents. The findings suggest a role for Ca2+/calmodulin-dependent protein kinase in Ca2+ modulation of proline transporters, and suggest that transport and substrate-gated currents can be regulated independently. 37. •

Ingram SL, Amara SG: Arachidonic acid stimulates a novel cocaine-sensitive cation conductance associated with the human dopamine transporter. J Neurosci 2000, 20:550-557. Arachidonic acid applied to Xenopus oocytes expressing human DAT induces a nonselective cation conductance yielding currents as much as 50 times greater than that seen in response to DA. Previously identified DAT currents do not appear to be amplified; rather, a novel conducting state seems to have been induced. Whether this behavior is exhibited by other amine transporters is unknown, but at minimum it defines a new paradigm for amplified analysis of DAT behavior. If these currents occur in vivo, they suggest an additional mode of arachidonic acid signaling that could be supported by DAT proteins. Because DA potentiates the current observed with arachionic acid, such a conducting state may provide for convergent presynaptic signaling mediated by DA release and arachidonate production. 38. Kantor L, Gnegy ME: Protein kinase C inhibitors block •• amphetamine-mediated dopamine release in rat striatal slices. J Pharmacol Exp Ther 1998, 284:592-598. Amphetamine-stimulated DA release in rat striatum is found to be sensitive to the prior application of PKC antagonists. Three different PKC inhibitors completely blocked amphetamine-induced DA release mediated by DAT with only modest effects on DA influx. This study provides evidence that influx and efflux can be regulated separately and that regulation independent of transporter trafficking may be controlled by PKC-dependent phosphorylation. 39. Browman KE, Kantor L, Richardson S, Badiani A, Robinson TE, • Gnegy ME: Injection of the protein kinase C inhibitor Ro31-8220 into the nucleus accumbens attenuates the acute response to amphetamine: tissue and behavioral studies. Brain Res 1998, 814:112-119. This study provides an in vivo correlate to the group’s in vitro efflux studies [38••], wherein tonic PKC activity in the rat nucleus accumbens is suggested to support amphetamine-induced DA efflux through DATs. PKC antagonists also attentuate the locomotor response to amphetamine. 40. Nirenberg MJ, Vaughan RA, Uhl GR, Kuhar MJ, Pickel VM: The dopamine transporter is localized to dendritic and axonal plasma membranes of nigrostriatal dopaminergic neurons. J Neurosci 1996, 16:436-447. 41. Tao-Cheng JH, Zhou FC: Differential polarization of serotonin • transporters in axons versus soma-dendrites: an immunogold electron microscopy study. Neuroscience 1999, 94:821-830. Using pre-embedding immunogold techniques, the authors describe the polarized expression of SERT protein distribution in serotonergic neurons. Immunoreactivity in the cell bodies and dendrites is cytoplasmic, whereas labeling in axons is membrane-associated. The extensive distribution of

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SERTs along axonal membranes supports a contribution of SERTs to 5-HT clearance beyond just the synaptic cleft. 42. Schroeter S, Apparsundaram S, Wiley RG, Miner LH, Sesack SR, • Blakely RD: Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter. J Comp Neurol 2000, 420:211-232. This study reports NET localization in the rat central and peripheral nervous systems, using light- and electron-microscopic immunocytochemical techniques. NET labeled neurons are seen in traditional noradrenergic neurons including the locus ceruleus. NET-positive fibers are found throughout the forebrain as well in peripherally innervated structures such as the vas deferens. Labeling in many axons appears puncate and colocalizes with dopamine beta hydroxylase, consistent with a role for NETs in clearing synaptic NE. NET-positive fibers are found near, but not colocalized with, phenylethanolamine-N-methyl transferase labeled fibers, suggesting that NETs may also play a role in clearance of released epinephrine. Significant intracellular NET expression is detected in the adrenal gland, in contrast to SERT expression in adrenal chromaffin cells, which is associated with the plasma membrane. 43. Schroeter S, Levey AI, Blakely RD: Polarized expression of the antidepressant-sensitive serotonin transporter in epinephrinesynthesizing chromaffin cells of the rat adrenal gland. Mol Cel Neurosci 1997, 9:170-184. 44. Perego C, Vanoni C, Villa A, Longhi R, Kaech SM, Frohli E, Hajnal A, •• Kim SK, Pietrini G: PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells. EMBO J 1999, 18:2384-2393. This is the first report of physical associations between PDZ domain proteins and members of the GAT1/NET gene family of Na+/Cl– coupled transporters. BGT-1, a transporter for betaine that polarizes to the basolateral domain in MDCK cells, is found to utilize carboxy-terminal sequences for basolateral stability rather than directed basolateral trafficking. The carboxyl terminus of BGT-1 is found to associate with LIN-7, a protein implicated in epithelial polarity from worms to mammals. The findings suggest that similar interactions may stabilize or recruit homologous transporters at membrane subdomains. 45. Lesch K-P, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Müller CR, Hamer DH, Murphy DL: Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996, 274:1527-1531. 46. Cook EH Jr, Courchesne R, Lord C, Cox NJ, Yan S, Lincoln A, Haas R, Courchesne E, Leventhal BL: Evidence of linkage between the serotonin transporter and autistic disorder. Mol Psych 1997, 2:247-250. 47.

Klauck SM, Poustka F, Benner A, Lesch KP, Poustka A: Serotonin transporter (5-HTT) gene variants associated with autism? Human Mol Genet 1997 6:2233-2238.

48. Greenberg BD, McMahon FJ, Murphy DL: Serotonin transporter candidate gene studies in affective disorders and personality: promises and potential pitfalls. Mol Psych 1998, 3:186-189. 49. Little KY, McLaughlin DP, Zhang L, Livermore CS, Dalack GW, • McFinton PR, DelProposto ZS, Hill E, Cassin BJ, Watson SJ, Cook EH: Cocaine, ethanol, and genotype effects on human midbrain serotonin transporter binding sites and mRNA levels. Am J Psych 1998, 155:207-213. Radioligand binding to SERT proteins in postmortem brain sections reveal a reduced density of SERTs in cocaine users. SERT mRNA expression and transporter density are found to be influenced by SERT promotor polymorphisms. However, SERT binding was elevated in those ethanol abusers bearing short promoter alleles. These findings are consistent with a role for SERT promoter polymorphisms in predicting the risk for substance abuse. 50. Cook EH, Stein MA, Krasowski MD, Cox NJ, Olkon DM, Kieffer JE, Leventhal BL: Association of attention-deficit disorder and the dopamine transporter gene. Am J Med Genet 1995, 56:993-998. 51. Daly G, Hawi Z, Fitzgerald M, Gill M: Mapping susceptibility loci in • attention deficit hyperactivity disorder: preferential transmission of parental alleles at DAT1, DBH and DRD5 to affected children. Mol Psych 1999, 4:192-196. This study reports preferential transmission of DAT alleles in ADHD, with the strongest findings reported in familial cases. The polymorphism tracked is not a coding mutation but may be linked with as yet unidentified coding alterations or noncoding mutations affecting DAT gene expression or, alternatively, affecting functional variants in a nearby gene.

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Signalling mechanisms

52. Gainetdinov RR, Wetsel WC, Jones SR, Levin ED, Jaber M, •• Caron MG: Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactivity. Science 1999, 283:397-401. Studies of DAT-deficient mice that are spontaneously hyperactive reveal a paradoxical calming effect of the psychostimulant methylphenidate (Ritalin), a pharmacologic phenotype that is observed in subjects with ADHD. Although methylphenidate is only a weak hSERT antagonist, in vivo studies in DAT-deficient mice support a role for serotonergic modulation in the compound’s calming actions. Cognitive impairments are also noted in DAT-deficient mice, although whether methylphenidate influences this phenotype is not reported. These studies encourage further exploration of DAT polymorphisms in human ADHD. 53. Shannon JR, Flattem NL, Jordan J, Jacob G, Black BK, Biaggioni I, •• Blakely RD, Robertson D: Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. New Engl J Med 2000, 342:541-549. This is the first report of coding variants in a biogenic amine transporter associated with a physiologic disturbance. Subjects are identified as heterozygous for a single nucleotide polymorphism (SNP) that results in the switch of a highly conserved TMD9 alanine residue to proline. The mutant NET is shown to be nonfunctional in heterologous expression systems. Phenotypes of elevated plasma NE, reduced DHPG (dihydroxyphenylglycol) production and tachycardia on standing are found to segregate with the A457P allele.

54. Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Lane CR, • Lim EP, Kalayanaraman N, Nemesh J et al.: Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nat Genet 1999, 22:231-238. A search for SNPs in a large number of neuronal genes reveals coding variations of potential physiologic relevance in several members of the GAT/NET family of Na+/Cl– coupled neurotransmitter transporters. 55. Halushka MK, Fan JB, Bentley K, Hsie L, Shen N, Weder A, Cooper R, • Lipshutz R, Chakravarti A: Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet 1999, 22:239-247. A study analogous to that of Cargill et al. [54•] focuses on blood pressure regulation and reveals multiple coding variants in the human NET gene. Although association studies remain to be conducted linking these coding variants to blood pressure, they augment significantly the number of known allelic variants of NETs and offer new opportunities to link transporter gene variation to cardiovascular traits. 56. Xu F, Gainetdinov RR, Wetsel WC, Jones SR, Bohn LM, Miller GW, •• Wang YM, Caron MG: Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat Neurosci 2000, 3:465-471. Xu and coworkers show that NET knockout mice have a diminished clearance capacity for NE and exhibit compensatory changes in dopaminergic signalling. See Update for further details.