Human phenotypes and animal knockout models of genetic autonomic disorders

Norepinephrine (NE) is a crucial neurotransmitter involved in autonomic regulation of blood pressure. Dopamine -hydroxylase (DBH), the norepinephrine transporter (NET), and the vesicular monoamine transporter subtype 2 catalyze intracellular NE biosynthesis, NE reuptake from the synapse, and vesicular transport, respectively. Genetic disorders in humans have been identified that render DBH, and the NET dysfunctional and result in cardiovascular and neurological abnormalities. Vesicular monoamine transporter subtype 2 (VMAT2) activity protects against neurotoxins, and reduced VMAT2 expression is implicated in drug addiction. Further investigation of the consequences of these genetic abnormalities has been achieved by the construction of mice strains deficient in the genes encoding DBH, NET, and VMAT2.     

Expression and Function of Variants of Human Catecholamine Transporters Lacking the Fifth Transmembrane Region Encoded by Exon 6

Background

The transporters for dopamine (DAT) and norepinephrine (NET) are members of the Na⁺- and Cl⁻-dependent neurotransmitter transporter family SLC6. There is a line of evidence that alternative splicing results in several isoforms of neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitter reuptake system has not been fully elucidated.

Methodology/Principal Findings

We found novel isoforms of human DAT and NET produced by alternative splicing in human blood cells (DAT) and placenta (NET), both of which lacked the region encoded by exon 6. RT-PCR analyses showed a difference in expression between the full length (FL) and truncated isoforms in the brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous expression of the FL but not truncated isoforms of DAT and NET in COS-7 cells revealed transport activity. However, immunocytochemistry with confocal microscopy and a cell surface biotinylation assay demonstrated that the truncated as well as FL isoform was expressed at least in part in the plasma membrane at the cell surface, although the truncated DAT was distributed to the cell surface slower than FL DAT. A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary. Co-expression of the FL isoform with the truncated isoform in COS-7 cells resulted in a reduced uptake of substrates, indicating a dominant negative effect of the variant. Furthermore, an immunoprecipitation assay revealed physical interaction between the FL and truncated isoforms.

Conclusions/Significance

The unique expression and function and the proposed membrane topology of the variants suggest the importance of isoforms of catecholamine transporters in monoaminergic signaling in the brain and peripheral tissues.     

C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.     

The role of external loop regions in serotonin transport. Loop scanning mutagenesis of the serotonin transporter external domain

Chimeric transporters were constructed in which the predicted external loops of the serotonin transporter (SERT) were replaced one at a time with a corresponding sequence from the norepinephrine transporter (NET). All of the chimeric transporters were expressed at levels equal to or greater than those of wild type SERT, but the transport and binding activity of the mutants varied greatly. In particular, mutants in which the NET sequence replaced external loops 4 or 6 of SERT had transport activity 5% or less than that of wild type, and the loop 5 replacement was essentially inactive. In some of these mutants, binding of a high affinity cocaine analog was less affected than transport, suggesting that the mutation had less effect on the initial binding steps in transport than on subsequent conformational changes. The more severely affected mutants also displayed an altered response to Na(+). In contrast to the dramatic reduction in transport and binding, the specificity of ligand binding was essentially unchanged. Chimeric transporters did not gain affinity for dopamine, a NET substrate, or desipramine, an inhibitor, at the expense of affinity for serotonin or paroxetine, a selective SERT inhibitor. The results suggest that external loops are not the primary determinants of substrate and inhibitor binding sites. However, they are not merely passive structures connecting transmembrane segments but rather active elements responsible for maintaining the stability and conformational flexibility of the transporter.     

Extracellular Norepinephrine Clearance by the Norepinephrine Transporter Is Required for Skeletal Homeostasis

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.     

Structural requirements for 2,4- and 3,6-disubstituted pyran biomimetics of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine compounds to interact with monoamine transporters

In our effort to delineate novel pharmacophoric configuration of bioisosteric pyran versions of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine derivatives in interacting with the monoamine transporter, further structure-activity relationship study was carried out. Both cis and trans 2,4- and 3,6-disubstituted derivatives were synthesized to determine the positional importance of N-substitution on affinity for monoamine transporters, that is the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET) in rat brain. For that purpose, the potency of compounds was determined in competing for the binding of [(3)H]WIN 35,428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]DA by DAT. Our binding results demonstrated potency in 3,6-disubstituted derivatives while 2,4-disubstituted derivatives failed to exhibit any appreciable binding affinity. Further structural exploration of the exocyclic N-atom in 3,6-disubstituted derivatives produced compounds potent at both DAT and NET. Compounds 16h and 16o with hydroxyl and amino groups in the phenyl moiety of the benzyl group produced the highest activity for the NET. In this regard, compound 16e with a methoxy substituent produced weak affinity at NET, which upon conversion into a hydroxyl functionality as in 16h produced potent affinity for the NET. Various indole derivatives displayed different interactions; the 5-substituted indole derivative 16n exerted potent affinity for NET, confirming the bioisosteric equivalence between this indole moiety and the phenyl-4-hydroxy group in 16h.     

Effects of desipramine treatment on norepinephrine transporter gene expression in the cultured SK-N-BE(2)M17 cells and rat brain tissue

The antidepressant desipramine (DMI) is a selective inhibitor of norepinephrine (NE) transport that down-regulates the norepinephrine transporter (NET) protein in a concentration- and time-dependent manner in vitro. In this study, possible regulatory effects of DMI on NET mRNA and protein levels were investigated with the NET-expressing SK-N-BE(2)M17 cell line and rat brain tissue. Northern blot analysis showed that incubation of the cultured cells with DMI (5-500 nm) for 3 days reduced levels of NET mRNA in both its 5.8-kb (by up to 58%) and 3.6-kb forms (to 68%), whereas incubation for 14 days increased both levels (to 40% and 100%) in a concentration-dependent manner. In contrast, NET protein levels decreased after 3-14 days of exposure of the cells to DMI, as determined by western blotting. The in vitro findings were supported by in vivo treatment of rats with DMI. Thus, in situ hybridization demonstrated initially decreased, and later increased, NET mRNA levels in locus coeruleus (LC) tissue of rats treated with DMI; whereas NET protein levels in the LC were reduced after 14 days, but unchanged after three daily DMI treatments. Thus, DMI had similar effects on NET expression in vitro and in vivo, with opposite changes in NET mRNA and protein levels, suggesting that the regulatory mechanisms involved are complex and non-congruent.     

Inhibition of nonexocytotic norepinephrine release by desipramine reduces myocardial infarction size

During myocardial ischemia, a substantial accumulation of norepinephrine occurs in the ischemic zone due to a local nonexocytotic release of norepinephrine. Norepinephrine release is driven by the neuronal monoamine transporter (NET), which reverses its usual transmembrane transport direction. We investigated whether this local accumulation of norepinephrine contributes to irreversible myocardial injury in an in vivo model of myocardial infarction. Male, anaesthetized Wistar rats were subjected to 30 min coronary occlusion and subsequent 120 min reperfusion. Five minutes prior to coronary occlusion, the NET inhibitor desipramine was administered intravenously. Infarct size (IS) was determined by TTC-staining and was related to the area at risk (AAR). The influence of desipramine on cardiac norepinephrine release was investigated in isolated perfused hearts with 30 min of regional ischemia. Norepinephrine was measured in the effluent from the hearts by HPLC and electrochemical detection. Desipramine (0.1-0.8 mg/kg) dose-dependently reduced infarct size (IS/AAR) from 0.54 to 0.21 and suppressed postischemic norepinephrine release from 245 to 108 pg/mL. In summary, the data indicate that nonexocytotic release of norepinephrine in myocardial ischemia exaggerates acute ischemic damage, because suppression of ischemia-induced release of norepinephrine by the tricyclic antidepressant desipramine effectively reduces infarct size in an in vivo model of myocardial ischemia.     

Inhibition of the norepinephrine transporter by the venom peptide chi-MrIA. Site of action,Na+ dependence,and structure-activity relationship

chi-Conopeptide MrIA (chi-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether chi-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 microM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by chi-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by chi-MrIA was competitive in nature. It has previously been demonstrated that chi-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for chi-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of chi-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, chi-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of chi-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of chi-MrIA and a His in loop 2 played a dominant role in the interaction between chi-MrIA and the NET. H alpha chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of chi-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.     

N-Linked Oligosaccharides Are Required for Cell Surface Expression of the Norepinephrine Transporter but Do Not Influence Substrate or Inhibitor Recognition

Abstract: The contribution of N-linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site-directed mutagenesis to inactivate the two most carboxy-terminal (NQQ mutant) or all three (QQQ mutant) sites for N -glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50-kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order V _max of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild-type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N-linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.     

The Role of Cysteines and Histidins of the Norepinephrine Transporter

The thiol reagent N-ethylmaleimide (NEM) is known to inhibit irreversibly ligand binding by the norepinephrine transporter (NET), while the simultaneous presence of NET substrates or ligands protects from this inhibition. Therefore, cysteine residues located within the substrate binding pocket of the NET were assumed to play an important role in ligand binding. To examine which (if any) of the 10 cysteines (Cys) of the human (h) NET might be involved in transport and/or binding function, we mutated all hNET cysteines to alanine. Using transfected HEK293 cells we studied NEM effects on the hNET with respect to [³H]nisoxetine binding. Two cysteines (Cys176 and Cys185) within the extracellular loop of the NET have been proposed to form a disulfide bond. We could demonstrate that this is of crucial importance as corresponding hNET mutants, in which these cysteines have been replaced, showed a lack of plasma membrane expression. However, due to their oxidized state in the native NET protein, Cys176 and Cys185 may not be targets for NEM. All other Cys-to-Ala hNET mutants were fully active and showed no change in inhibition of [³H]nisoxetine binding by NEM. These observations clearly exclude cysteines as being involved in hNET ligand binding. Since NEM also interacts with histidin (His), we mutated all 13 histidins of the hNET to alanine and examined the NET mutants in functional and binding assays. His222 within the large extracellular loop of the transporter was identified as an interaction partner of NEM since in the corresponding hNET mutant NEM exhibited a significantly reduced inhibitory potency. Furthermore, we could show that histidins in position 296, 370 and 372 are important for nisoxetine binding, while His220, 441, 598 and 599 are crucial for plasma membrane expression of the hNET.     

chi-Conotoxin and tricyclic antidepressant interactions at the norepinephrine transporter define a new transporter model

Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide chi-MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids (Leu(469) and Glu(382)) that affected the affinity of chi-MrIA to inhibit [(3)H]NE uptake through human NET. Residues that increased the K(d) of a tricyclic antidepressant (nisoxetine) were also identified (Phe(207), Ser(225), His(296), Thr(381), and Asp(473)). Phe(207), Ser(225), His(296), and Thr(381) also affected the rate of NE transport without affecting NE K(m). In a new model of NET constructed from the bLeuT crystal structure, chi-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors.     

The NH2-terminus of Norepinephrine Transporter Contains a Basolateral Localization Signal for Epithelial Cells

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT-NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH(2)-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH(2)-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH(2)-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.     

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.     

Altered Reward Circuitry in the Norepinephrine Transporter Knockout Mouse

Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn²⁺ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of more robust connectivity in the frontal portion of the reward circuit of the DAT and NET knockout mice compared to the SERT knockout mice.     

Synthesis and biological evaluation of a series of novel N- or O-fluoroalkyl derivatives of tropane: potential positron emission tomography (PET) imaging agents for the dopamine transporter.

A series of novel fluoroalkyl-containing tropane derivatives was synthesized, and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined via competitive binding assays. Among these derivatives, the fluoropropyl ester of beta-CIT (19), the fluoroethyl ester of beta-CIT (20), the N-fluoropropyl derivative of beta-CBT (12), and the fluoropropyl ester of beta-CMT (18) displayed higher affinity and greater selectivity for the DAT versus SERT and NET than FP-CIT, which indicates that they are attractive candidates for the development of (18)F-labeled PET imaging agents for the DAT.     

Discovery of a potent, selective, and less flexible selective norepinephrine reuptake inhibitor (sNRI)

The design, synthesis, and SAR of a series of ring-constrained norepinephrine reuptake inhibitors are described. A substantially rigid inhibitor with potent functional activity at the transporter (IC₅₀ = 8 nM) was used to develop a model for the distance and orientation of key features necessary for interaction with the norepinephrine transporter (NET).     

Substrate binding stoichiometry and kinetics of the norepinephrine transporter

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.     

Synthesis of some tropane derivatives of anticipated activity on the reuptake of norepinephrine and/or serotonin

A variety of tropane derivatives 14a–g were prepared via the reaction of the alcohol analogs 12a and 12b with substituted fluorobenzenes 13a–f. The prepared compounds were tested for their activity and selectivity toward the norepinephrine transporter (NET) and serotonin transporter (SERT) using yohimbine-induced mortality and 5-hydroxytryptophan-induced neurotoxicity in mice, respectively. All the tested compounds were found to be NE and 5-HT reuptake inhibitors except 14d which exhibited selective 5-HT reuptake inhibition activity.     

Design, synthesis, and preliminary SAR study of 3- and 6-side-chain-extended tetrahydro-pyran analogues of cis- and trans-(6-benzhydryl-tetrahydropyran-3-yl)-benzylamine

In our effort to further understand interaction of novel pyran derivatives with monoamine transporters, we have designed, synthesized, and biologically characterized side-chain-extended derivatives of our earlier developed cis- and trans-(6-benzhydryl-tetrahydro-pyran-3-yl)-benzylamine derivatives. Both 3- and 6-position extensions were explored. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding affinity at the DAT by their ability to inhibit binding of [(3)H]WIN 35, 428. The results indicated that extension at the 3-position resulted in loss of activity compared to the original compound I. On the other hand, extension at the 6-position resulted in improvement of activity in the compound cis-12 by 2-fold over the parent compound I indicating favorable interaction. In addition, two glycoside derivatives were designed, synthesized, and biologically characterized. The glycosidic trans-isomer 24 exhibited highest potency for the NET in the current series of compounds.