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1.  Residue Ile89 in Human Plasma Membrane Monoamine Transporter influences its Organic Cation Transport Activity and Sensitivity to Inhibition by Dilazep 
Biochemical Pharmacology  2012;84(3):383-390.
Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin-Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (Vmax) with no significant change in apparent binding affinity (Km) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT’s sensitivity to dilazep by 2.5 fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.
doi:10.1016/j.bcp.2012.04.018
PMCID: PMC3372611  PMID: 22562044
PMAT; SLC29 family; ENT4; Dilazep; Dipyridamole
2.  Identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a Protein Receptor for Botulinum Neurotoxin Serotype A (BoNT/A) 
PLoS Pathogens  2013;9(5):e1003369.
Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.
Author Summary
Botulinum neurotoxin serotype A (BoNT/A) is one of seven neurotoxins (BoNT/A-G), produced by the bacteria Clostridium botulinum that are both poisons and versatile therapeutics. These toxins enter motor neurons where they prevent the release of acetylcholine at the neuromuscular junction. The specific uptake of BoNT/A across the neuronal cell membrane is dependent on specific receptor interactions. Binding to high density ganglioside GT1b mediates the initial binding step and via a low affinity interaction concentrates BoNT/A on the cell surface. Once anchored in the membrane, lateral movements within the plasma membrane facilitate intermolecular interactions of BoNT/A with additional lower density but higher affinity protein receptors. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A. We show that BoNT/A binds to FGFR3 with high affinity and functions as an agonist ligand for FGFR3. The identification of this novel receptor for BoNT/A represents an important advance in the understanding of the mechanism of action of BoNT/A, especially on the initial steps of neuronal uptake, and can be the basis for the development of new specific countermeasures and new BoNT/A-based therapeutics.
Highlights
▸ Recombinant HC/A binds to the two extra-cellular loops of FGFR3b with a KD∼15 nM ▸ Recombinant HC/A acts as an agonist ligand for FGFR3 ▸The level of BoNT/A uptake is dependent on FGFR3 expression ▸ FGFR3 is expressed in motor nerve terminals
doi:10.1371/journal.ppat.1003369
PMCID: PMC3656097  PMID: 23696738
3.  A Pilot Pharmacologic Biomarker Study of Busulfan and Fludarabine in Hematopoietic Cell Transplant Recipients 
Purpose
Sixteen patients diagnosed with various hematologic malignancies participated in a phase II study evaluating the addition of rabbit antithymocyte globulin (rATG, Thymoglobulin®) to the hematopoietic cell transplant (HCT) conditioning regimen of IV fludarabine monophosphate (fludarabine) and targeted intravenous (IV) busulfan (fludarabine/Tbusulfan). Our goal was to evaluate pharmacologic biomarkers pertinent to both medications in these patients.
Methods
We characterized the interpatient variability of pharmacologic biomarkers relevant to busulfan, specifically busulfan concentration at steady state (Css), and fludarabine, specifically F-ara-A area under the curve (AUC) and fludarabine triphosphate (F-ara-ATP) intracellular accumulation and concentration in separate CD4+ and CD8+ T-lymphocyte populations.
Results
Acute and chronic graft versus host disease (GvHD) occurred in 11 patients and one patient, respectively. Four patients died before day +100 of non-relapse causes, which met the protocol stopping guidelines. The cumulative incidence of relapse was 25% at three year post-HCT. Interpatient variability in the busulfan- and fludarabine-relevant pharmacologic biomarkers was 2.1- to 2.7-fold. F-ara-A AUC and accumulated F-ara-ATP in CD8+ cells had the highest hazard ratio for non-relapse mortality and overall survival, respectively. However, neither achieved statistical significance.
Conclusions
The low rates of GvHD, particularly in its chronic form, were encouraging and further biomarker studies are warranted to optimize the fludarabine/Tbusulfan/rATG conditioning regimen.
doi:10.1007/s00280-011-1736-3
PMCID: PMC3280823  PMID: 21909959
Busulfan; fludarabine; hematopoietic cell transplant; biomarkers; therapeutic drug monitoring; pharmacokinetics
4.  Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay 
PLoS ONE  2012;7(11):e49516.
Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.
doi:10.1371/journal.pone.0049516
PMCID: PMC3504020  PMID: 23185348
5.  Expression Profiling of Solute Carrier Gene Families at the Blood-CSF Barrier 
The choroid plexus (CP) is a highly vascularized tissue in the brain ventricles and acts as the blood-cerebrospinal fluid (CSF) barrier (BCSFB). A main function of the CP is to secrete CSF, which is accomplished by active transport of small ions and water from the blood side to the CSF side. The CP also supplies the brain with certain nutrients, hormones, and metal ions, while removing metabolites and xenobiotics from the CSF. Numerous membrane transporters are expressed in the CP in order to facilitate the solute exchange between the blood and the CSF. The solute carrier (SLC) superfamily represents a major class of transporters in the CP that constitutes the molecular mechanisms for CP function. Recently, we systematically and quantitatively examined Slc gene expression in 20 anatomically comprehensive brain areas in the adult mouse brain using high-quality in situ hybridization data generated by the Allen Brain Atlas. Here we focus our analysis on Slc gene expression at the BCSFB using previously obtained data. Of the 252 Slc genes present in the mouse brain, 202 Slc genes were found at detectable levels in the CP. Unsupervised hierarchical cluster analysis showed that the CP Slc gene expression pattern is substantially different from the other 19 analyzed brain regions. The majority of the Slc genes in the CP are expressed at low to moderate levels, whereas 28 Slc genes are present in the CP at the highest levels. These highly expressed Slc genes encode transporters involved in CSF secretion, energy production, and transport of nutrients, hormones, neurotransmitters, sulfate, and metal ions. In this review, the functional characteristics and potential importance of these Slc transporters in the CP are discussed, with particular emphasis on their localization and physiological functions at the BCSFB.
doi:10.3389/fphar.2012.00154
PMCID: PMC3426838  PMID: 22936914
choroid plexus; blood-cerebrospinal fluid barrier; BCSFB; solute carriers; CSF; transporters; Slc gene; Allen Brain Atlas
6.  Effect of human beta-defensin-3 on head and neck cancer cell migration using micro-fabricated cell islands 
Head & Neck Oncology  2012;4:41.
Background
To examine the effect of the natural antimicrobial peptide human β-defensin-3 (hBD-3), on the migration of a head and neck cancer cell line in vitro using microfabrication and soft-lithographic techniques.
Methods
TR146 cancer cells were seeded in Petri dishes with microfabricated wells for cell migration assays. Total 54 cell islands were used of various shape and size and experimental media type. Cell migration assays were analyzed in six group media: Dulbecco’s modified medium (DMEM); DMEM with vascular endothelial growth factor (VEGF); Conditioned media of human embryonic kidney cells (HEK 239) expressing hBD-3 via transfected cloned pcDNA3 as CM/hBD-3; CM/hBD-3 + VEGF; conditioned medium from non-transfected HEK 239 (not expressing hBD-3) as control (CM); and the last group was CM + VEGF. Cell islands were circular or square and varied in size (0.25 mm2, 0.125 mm2, and 0.0625 mm2). Cell islands were imaged at t = 0 h, 3 h, 6 h, and 24 h.
Results
The results show cancer cell islands that originally were smaller had higher migration indices. There was no difference of MIs between circular and square cell islands. MIs at the end point were significantly different among the groups except between CM and CM-hBD-3+ VEGF.
Conclusions
VEGF enhanced cancer cell migration. The combination of DMEM and VEGF showed a synergistic effect on this phenomenon of cancer cell migration. Conditioned medium with hBD-3 suppressed cancer cell migration. hBD-3 suppressed VEGF enhancement of TR146 cancer cell migration.
doi:10.1186/1758-3284-4-41
PMCID: PMC3448511  PMID: 22742448
Head and neck cancer; Cancer cell migration; Human beta defensin; Migration index; Micro-fabricated cell islands
7.  Evidence for CRHR1 in multiple sclerosis using supervised machine learning and meta-analysis in 12 566 individuals 
Human Molecular Genetics  2010;19(21):4286-4295.
The primary genetic risk factor in multiple sclerosis (MS) is the HLA-DRB1*1501 allele; however, much of the remaining genetic contribution to MS has yet to be elucidated. Several lines of evidence support a role for neuroendocrine system involvement in autoimmunity which may, in part, be genetically determined. Here, we comprehensively investigated variation within eight candidate hypothalamic–pituitary–adrenal (HPA) axis genes and susceptibility to MS. A total of 326 SNPs were investigated in a discovery dataset of 1343 MS cases and 1379 healthy controls of European ancestry using a multi-analytical strategy. Random Forests, a supervised machine-learning algorithm, identified eight intronic SNPs within the corticotrophin-releasing hormone receptor 1 or CRHR1 locus on 17q21.31 as important predictors of MS. On the basis of univariate analyses, six CRHR1 variants were associated with decreased risk for disease following a conservative correction for multiple tests. Independent replication was observed for CRHR1 in a large meta-analysis comprising 2624 MS cases and 7220 healthy controls of European ancestry. Results from a combined meta-analysis of all 3967 MS cases and 8599 controls provide strong evidence for the involvement of CRHR1 in MS. The strongest association was observed for rs242936 (OR = 0.82, 95% CI = 0.74–0.90, P = 9.7 × 10−5). Replicated CRHR1 variants appear to exist on a single associated haplotype. Further investigation of mechanisms involved in HPA axis regulation and response to stress in MS pathogenesis is warranted.
doi:10.1093/hmg/ddq328
PMCID: PMC2951862  PMID: 20699326
8.  Tyrosine 112 is Essential for Organic Cation Transport by the Plasma Membrane Monoamine Transporter 
Biochemistry  2010;49(36):7839-7846.
Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter in the solute carrier 29 (SLC29) family. Previous studies suggested that the major substrate recognition site is located within TM1-6 and PMAT interaction with organic cations may involve aromatic residues. In this study, we analyzed the role of tyrosine and tryptophan residues located within TM1-6 with the goal to identify potential residues involved in substrate recognition and translocation. The six Tyr and one Trp residues in this region were each mutated to alanine followed by analysis of these mutants’ membrane localization and transport activity towards 1-methyl-4-phenylpyridinium (MPP+), serotonin (5-HT) and dopamine. Two mutants, Y85A and Y112A, showed normal cell surface expression but lost their transport activity towards organic cations. At position Y85, aromatic substitution with Phe or Trp fully restored organic cation transport activity. Interestingly, at position Y112, Phe substitution is not allowed. Trp substitution at Y112 partially restored transport activity towards 5-HT and dopamine, but severely impaired MPP+ transport. Detailed kinetic analysis revealed that Trp substitution at Y85 and Y112 affected apparent binding affinity (Km) and maximal transport velocity (Vmax) in a substrate-dependent manner. Together these data suggest that Y85 and Y112 are important molecular determinants for PMAT function, and Y112 is indispensable for optimal interaction with organic cation substrates. Our analysis also suggested the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.
doi:10.1021/bi100560q
PMCID: PMC2995015  PMID: 20687515
9.  Modeling the cumulative genetic risk for multiple sclerosis from genome-wide association data 
Genome Medicine  2011;3(1):3.
Background
Multiple sclerosis (MS) is the most common cause of chronic neurologic disability beginning in early to middle adult life. Results from recent genome-wide association studies (GWAS) have substantially lengthened the list of disease loci and provide convincing evidence supporting a multifactorial and polygenic model of inheritance. Nevertheless, the knowledge of MS genetics remains incomplete, with many risk alleles still to be revealed.
Methods
We used a discovery GWAS dataset (8,844 samples, 2,124 cases and 6,720 controls) and a multi-step logistic regression protocol to identify novel genetic associations. The emerging genetic profile included 350 independent markers and was used to calculate and estimate the cumulative genetic risk in an independent validation dataset (3,606 samples). Analysis of covariance (ANCOVA) was implemented to compare clinical characteristics of individuals with various degrees of genetic risk. Gene ontology and pathway enrichment analysis was done using the DAVID functional annotation tool, the GO Tree Machine, and the Pathway-Express profiling tool.
Results
In the discovery dataset, the median cumulative genetic risk (P-Hat) was 0.903 and 0.007 in the case and control groups, respectively, together with 79.9% classification sensitivity and 95.8% specificity. The identified profile shows a significant enrichment of genes involved in the immune response, cell adhesion, cell communication/signaling, nervous system development, and neuronal signaling, including ionotropic glutamate receptors, which have been implicated in the pathological mechanism driving neurodegeneration. In the validation dataset, the median cumulative genetic risk was 0.59 and 0.32 in the case and control groups, respectively, with classification sensitivity 62.3% and specificity 75.9%. No differences in disease progression or T2-lesion volumes were observed among four levels of predicted genetic risk groups (high, medium, low, misclassified). On the other hand, a significant difference (F = 2.75, P = 0.04) was detected for age of disease onset between the affected misclassified as controls (mean = 36 years) and the other three groups (high, 33.5 years; medium, 33.4 years; low, 33.1 years).
Conclusions
The results are consistent with the polygenic model of inheritance. The cumulative genetic risk established using currently available genome-wide association data provides important insights into disease heterogeneity and completeness of current knowledge in MS genetics.
doi:10.1186/gm217
PMCID: PMC3092088  PMID: 21244703
10.  Multiple sclerosis susceptibility alleles in African Americans 
Genes and immunity  2009;11(4):343-350.
Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by complex genetics and multifaceted gene-environment interactions. Compared to whites, African Americans have a lower risk for developing MS, but African Americans with MS have a greater risk of disability. These differences between African Americans and whites may represent differences in genetic susceptibility and/or environmental factors. SNPs from 12 candidate genes have recently been identified and validated with MS risk in white populations. We performed a replication study using 918 cases and 656 unrelated controls to test whether these candidate genes are also associated with MS risk in African Americans. CD6, CLEC16a, EVI5, GPC5, and TYK2 contained SNPs that are associated with MS risk in the African American dataset. EVI5 showed the strongest association outside the MHC (rs10735781, OR = 1.233, 95% CI = 1.06–1.43, P value = 0.006). In addition, RGS1 appears to affect age of onset whereas TNFRSF1A appears to be associated with disease progression. None of the tested variants showed results that were statistically in-consistent with the effects established in whites. The results are consistent with shared disease genetic mechanisms among individuals of European and African ancestry.
doi:10.1038/gene.2009.81
PMCID: PMC2880217  PMID: 19865102
11.  INTRACELLULAR DISPOSITION OF FLUDARABINE TRIPHOSPHATE IN HUMAN NATURAL KILLER CELLS 
Purpose
Fludarabine is a key component of several reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Shortly after reduced-intensity conditioning, the percent of donor natural killer (NK) cells has been associated with progression-free survival. Insufficient suppression of the recipient’s NK cells by fludarabine may lead to lower donor chimerism; however, the effect of fludarabine upon NK cells is poorly understood. Thus, in purified human NK cells we evaluated the uptake and activation of fludarabine to its active metabolite, fludarabine triphosphate (F-ara-ATP), and assessed the degree of interindividual variability in F-ara-ATP accumulation.
Methods
Intracellular F-ara-ATP was measured in purified NK cells isolated from healthy volunteers (n = 6) after ex vivo exposure to fludarabine. Gene expression levels of the relevant transporters and enzymes involved in fludarabine uptake and activation were also measured in these cells.
Results
F-ara-ATP accumulation (mean ± s.d.) was 6.00 ± 3.67 pmol/1×106 cells/4 hours, comparable to average levels previously observed in CD4+ and CD8+ T-lymphocytes. We observed considerable variability in F-ara-ATP accumulation and mRNA expression of transporters and enzymes relevant to F-ara-ATP accumulation in NK cells from different healthy volunteers.
Conclusions
Human NK cells have the ability to form F-ara-ATP intracellularly and large interindividual variability was observed in healthy volunteers. Further studies are needed to evaluate whether F-ara-ATP accumulation in NK cells are associated with apoptosis and clinical outcomes.
doi:10.1007/s00280-008-0829-0
PMCID: PMC2648818  PMID: 18781301
fludarabine; fludarabine triphosphate; natural killer cells; hematopoietic cell transplantation; chimerism; nucleoside transporters
12.  Effects of Cranberry Juice on Pharmacokinetics of β-Lactam Antibiotics following Oral Administration▿  
Cranberry juice consumption is often recommended along with low-dose oral antibiotics for prophylaxis for recurrent urinary tract infection (UTI). Because multiple membrane transporters are involved in the intestinal absorption and renal excretion of β-lactam antibiotics, we evaluated the potential risk of pharmacokinetic interactions between cranberry juice and the β-lactams amoxicillin (amoxicilline) and cefaclor. The amoxicillin-cranberry juice interaction was investigated in 18 healthy women who received on four separate occasions a single oral test dose of amoxicillin at 500 mg and 2 g with or without cranberry juice cocktail (8 oz) according to a crossover design. A parallel cefaclor-cranberry juice interaction study was also conducted in which 500 mg cefaclor was administered with or without cranberry juice cocktail (12 oz). Data were analyzed by noncompartmental methods and nonlinear mixed-effects compartmental modeling. We conclude that the concurrent use of cranberry juice has no significant effect on the extent of oral absorption or the renal clearance of amoxicillin and cefaclor. However, delays in the absorption of amoxicillin and cefaclor were observed. These results suggest that the use of cranberry juice at usual quantities as prophylaxis for UTI is not likely to alter the pharmacokinetics of these two oral antibiotics.
doi:10.1128/AAC.00774-08
PMCID: PMC2704661  PMID: 19398645
13.  Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci 
Nature genetics  2009;41(7):776-782.
We report the results of a meta-analysis of genome-wide association scans for multiple sclerosis (MS) susceptibility that includes 2,624 subjects with MS and 7,220 control subjects. Replication in an independent set of 2,215 subjects with MS and 2,116 control subjects validates new MS susceptibility loci at TNFRSF1A (combined P = 1.59 × 10−11), IRF8 (P = 3.73 × 10−9) and CD6 (P = 3.79 × 10−9). TNFRSF1A harbors two independent susceptibility alleles: rs1800693 is a common variant with modest effect (odds ratio = 1.2), whereas rs4149584 is a nonsynonymous coding polymorphism of low frequency but with stronger effect (allele frequency = 0.02; odds ratio = 1.6). We also report that the susceptibility allele near IRF8, which encodes a transcription factor known to function in type I interferon signaling, is associated with higher mRNA expression of interferon-response pathway genes in subjects with MS.
doi:10.1038/ng.401
PMCID: PMC2757648  PMID: 19525953
14.  Expression Profiling of the Solute Carrier (SLC) Gene Family in the Mouse Brain 
The solute carrier (Slc) superfamily is a major group of membrane transport proteins present in mammalian cells. While Slc transporters play essential and diverse roles in the central nervous system, the localization and function of the vast majority of Slc genes in the mammalian brain are largely unknown. Using high throughput in situ hybridization data generated by the Allen Brain Atlas, we systematically and quantitatively analyzed the spatial and cellular distribution of 307 Slc genes, which represent nearly 90% of presently known mouse Slc genes, in the adult C57BL/6J mouse brain. Our analysis showed that 252 (82%) of the 307 Slc genes are present in the brain, and a large proportion of these genes were detected at low to moderate expression levels. Evaluation of 20 anatomical brain subdivisions demonstrated a comparable level of Slc gene complexity, but significant difference in transcript enrichment. The distribution of the expressed Slc genes was diverse, ranging from near-ubiquitous to highly localized. Functional annotation in 20 brain regions, including the blood-brain and blood-cerebral spinal fluid (CSF) barriers, suggest major roles of Slc transporters in supporting brain energy utilization, neurotransmission, nutrient supply, and CSF production. Further, hierarchical cluster analysis revealed intricate Slc expression patterns associated with neuroanatomical organization. Our studies also revealed Slc genes present within defined brain microstructures and described the putative cell types expressing individual Slc genes. These results provide a useful resource for investigators to explore the roles of Slc genes in neurophysiological and pathological processes.
doi:10.1124/jpet.108.149831
PMCID: PMC2672879  PMID: 19179540
15.  Molecular Determinants of Substrate Selectivity of a Novel Organic Cation Transporter (PMAT) in the SLC29 Family* 
The Journal of biological chemistry  2006;282(5):3188-3195.
Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1–3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1–6 of PMAT and TM7–11 of hENT1 behaved like PMAT, transporting 1-methyl-4-phenylpyridinium (MPP+, an organic cation) but not uridine (a nucleoside), suggesting that TM1–6 contains critical domains responsible for substrate recognition. To identify residues important for the cation selectivity of PMAT, 10 negatively charged residues were chosen and substituted with alanine. Five of the alanine mutants retained PMAT activity, and four were non-functional due to impaired targeting to the plasma membrane. However, alanine substitution at Glu206 in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the charge at Glu206 (E206Q) resulted in loss of organic cation transport activity, whereas conserving the negative charge (E206D) restored transporter function. Interestingly, mutant E206Q, which possesses the equivalent residue in ENT1, gained uridine transport activity. Thr220, another residue in TM5, also showed an effect on PMAT activity. Helical wheel analysis of TM5 revealed a distinct amphipathic pattern with Glu206 and Thr220 clustered in the center of the hydrophilic face. In summary, our results suggest that Glu206 functions as a critical charge sensor for cationic substrates and TM5 forms part of the substrate permeation pathway in PMAT.
doi:10.1074/jbc.M609421200
PMCID: PMC2672960  PMID: 17121826
16.  Metformin Transport by a Newly Cloned Proton-Stimulated Organic Cation Transporter (Plasma Membrane Monoamine Transporter) Expressed in Human Intestine 
Metformin is a widely used oral antihyperglycemic drug for the treatment of type II diabetes mellitus. The intestinal absorption of metformin is dose-dependent and involves an active, saturable uptake process. Metformin has been shown to be transported by the human organic cation transporters 1 and 2 (hOCT1–2). We recently cloned and characterized a novel proton-activated organic cation transporter, plasma membrane monoamine transporter (PMAT). We previously showed that PMAT transports many classic organic cations (e.g., monoamine neurotransmitters, 1-methyl-4-phenylpyridinium) in a pH-dependent manner and its mRNA is expressed in multiple human tissues. The goal of this study is to investigate whether metformin is a substrate of PMAT and whether PMAT plays a role in the intestinal uptake of metformin. Using Madin-Darby canine kidney cells stably expressing human PMAT, we showed that metformin is avidly transported by PMAT, with an apparent affinity (Km = 1.32 mM) comparable to those reported for hOCT1–2. Interestingly, the concentration-velocity profile of PMAT-mediated metformin uptake is sigmoidal, with a Hill coefficient of 2.64. PMAT-mediated metformin transport is greatly stimulated by acidic pH, with the uptake rate being ~4-fold higher at pH 6.6 than at pH 7.4. Using a polyclonal antibody against PMAT, we showed that the PMAT protein (58 kDa) was expressed in human small intestine and concentrated on the tips of the mucosal epithelial layer. Taken together, our results suggest that PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorption of metformin and possibly other cationic drugs.
doi:10.1124/dmd.107.015495
PMCID: PMC2672958  PMID: 17600084
17.  Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells 
Plasma membrane monoamine transporter (PMAT) is a novel membrane transporter recently cloned and characterized in our laboratory. We previously demonstrated that PMAT functions as a polyspecific organic cation transporter and efficiently transports many organic cations such as monoamine neurotransmitters and 1-methyl-4-phenylpyridinium (MPP+). In this study, we explored the role of PMAT in the renal handling of organic cations. Using a polyclonal antibody generated toward the NH2-terminal 66 amino acid residues of human PMAT, we showed that the PMAT protein (~55 kDa) is expressed in the human kidney and is primarily targeted to the apical membranes when expressed in polarized Madin-Darby canine kidney (MDCK) cells. Using MDCK cells stably expressing human PMAT, we showed that PMAT-mediated MPP+ uptake is strongly dependent on extracellular pH. Lowering extracellular pH from 7.4 to 6.6 greatly stimulated PMAT-mediated MPP+ uptake, whereas elevating extracellular pH to 8.2 abolished transporter activity. Kinetic analysis revealed that the apparent Vmax at pH 6.6 is about fourfold higher than that at pH 7.4, whereas the apparent Km values were not statistically different at these two conditions. Under acidic conditions (pH 6.6), the proton ionophore, carbonyl cyanide p-trifluormethoxyphenylhydrazone, drastically reduced PMAT-mediated MPP+ uptake, suggesting that the stimulatory effect of proton may be due to transporter coupling with a proton gradient. Taken together, our data suggest that PMAT is expressed on the apical membranes of renal epithelial cells and may use luminal proton gradient to drive organic cation reabsorption in the kidney.
doi:10.1152/ajprenal.00302.2006
PMCID: PMC2672957  PMID: 17018840
organic cation; MPP+; apical membrane; equilibrative nucleoside transporter 4; monoamine neurotransmitters
18.  Pathway and network-based analysis of genome-wide association studies in multiple sclerosis 
Human Molecular Genetics  2009;18(11):2078-2090.
Genome-wide association studies (GWAS) testing several hundred thousand SNPs have been performed in multiple sclerosis (MS) and other complex diseases. Typically, the number of markers in which the evidence for association exceeds the genome-wide significance threshold is very small, and markers that do not exceed this threshold are generally neglected. Classical statistical analysis of these datasets in MS revealed genes with known immunological functions. However, many of the markers showing modest association may represent false negatives. We hypothesize that certain combinations of genes flagged by these markers can be identified if they belong to a common biological pathway. Here we conduct a pathway-oriented analysis of two GWAS in MS that takes into account all SNPs with nominal evidence of association (P < 0.05). Gene-wise P-values were superimposed on a human protein interaction network and searches were conducted to identify sub-networks containing a higher proportion of genes associated with MS than expected by chance. These sub-networks, and others generated at random as a control, were categorized for membership of biological pathways. GWAS from eight other diseases were analyzed to assess the specificity of the pathways identified. In the MS datasets, we identified sub-networks of genes from several immunological pathways including cell adhesion, communication and signaling. Remarkably, neural pathways, namely axon-guidance and synaptic potentiation, were also over-represented in MS. In addition to the immunological pathways previously identified, we report here for the first time the potential involvement of neural pathways in MS susceptibility.
doi:10.1093/hmg/ddp120
PMCID: PMC2678928  PMID: 19286671
19.  The Detection of Alkaline Phosphatase Using an Electrochemical Biosensor in a Single-Step Approach 
Sensors (Basel, Switzerland)  2009;9(11):8709-8721.
A one-step, single use, disposable Alkaline Phosphatase (ALP) biosensor has been developed. It is based on the detection of phenol produced by an ALP enzymatic reaction. It can operate at 25 °C in a pH 10 medium. It measures ALP of 0–300 IU/L. The permissible concentrations of glucose, ascorbic acid and urea without interference are 10 mM/L, 5 mg/L and 400 mg/L, respectively. Experimental results are compared to those obtained by spectrophotometric measurements in bovine serum. Excellent linearity between the biosensor outputs and the ALP concentrations exists. The agreement between the measurements of this biosensor and the spectrophotometer is also outstanding.
doi:10.3390/s91108709
PMCID: PMC3260609  PMID: 22291532
alkaline phosphatase; electrochemical detection; biosensor
20.  Adenosine Transporter ENT4 Is a Direct Target of EWS/WT1 Translocation Product and Is Highly Expressed in Desmoplastic Small Round Cell Tumor 
PLoS ONE  2008;3(6):e2353.
Background
Desmoplastic Small Round Cell Tumor (DSRCT) is a highly aggressive malignancy that affects mainly adolescents and young adults. A defining characteristic of DSRCT is a specific chromosomal translocation, t(11;22)(p13;q12), that fuses EWS with WT1, leading to a production of two isoforms of chimeric transcription factor, EWS/WT1(−KTS) and EWS/WT1(+KTS). The chimeric proteins are thought to play critical roles in various stages of oncogenesis through aberrant transcription of different genes, but only a few of these genes have been identified.
Methodology/Principal Findings
We report the identification of a new target of EWS/WT1, ENT4 (equilibrative nucleoside transporter 4) which encodes a pH-dependent adenosine transporter. ENT4 is transcriptionally activated by both isoforms of EWS/WT1 as evidenced by promoter-reporter and chromatin immunoprecipitation (ChIP) analyses. Furthermore, ENT4 is highly and specifically expressed in primary tumors of DSRCT as well as in a DSRCT cell line, JN-DSRCT-1. Treatment of JN-DSRCT-1 cells with adenosine analogs, such as 2-chloro-2′-deoxyadenosine (2-CdA), resulted in an increased cytotoxic response in dose- and pH-dependent manner.
Conclusions/Significance
Our detailed analyses of a novel target of EWS/WT1 in DSRCT reveal an insight into the oncogenic mechanism of EWS-fusion chromosomal translocation gene products and provide a new marker for DSRCT. Furthermore, identification of ENT4 as a highly expressed transcript in DSRCT may represent an attractive pathway for targeting chemotherapeutic drugs into DSRCT.
doi:10.1371/journal.pone.0002353
PMCID: PMC2394657  PMID: 18523561
21.  Evidence for Significant Contribution of a Newly Identified Monoamine Transporter (PMAT) to Serotonin Uptake in the Human Brain 
Biochemical pharmacology  2006;73(1):147-154.
The high affinity serotonin transporter (SERT) constitutes the principal pathway for removal of serotonin (5-HT) from extracellular fluid of brain, but evidence indicates that other transporters may also be involved in this process. We recently reported the cloning of a novel plasma membrane monoamine transporter (PMAT), which is abundantly expressed in the human brain and avidly transports 5-HT (J Biol Chem 279(48):50042–9, 2004) . In this study, we evaluated whether PMAT contributes to total human brain uptake of 5-HT using a hybrid depletion approach in Xenopus laevis oocytes. We also examined whether PMAT interacts with selective serotonin reuptake inhibitors (SSRIs) using MDCK cells stably expressing recombinant human PMAT. Microinjection of total human brain poly(A)+ mRNA into oocytes elicited ~2.5-3 fold increase in 5-HT uptake. Pre-hybridization of poly(A)+ mRNA with PMAT or SERT antisense oligonucleotides significantly reduced mRNA-induced 5-HT uptake. An additive inhibitory effect was observed when poly(A)+ mRNA was co-hybridized with both PMAT and SERT antisense oligonucleotides. In contrast, mRNA-induced 5-HT uptake was not affected by pre-hybridization with sense oligonucleotides. These data suggest that functional transcripts of PMAT are present in the human brain, and the PMAT transporter may be significantly involved in brain uptake of 5-HT. All five tested SSRIs inhibited PMAT with IC50 values ranging from 11-116 μM, which are much greater than clinically encountered concentrations, suggesting that PMAT activity is minimally affected by SSRI therapies.
doi:10.1016/j.bcp.2006.09.008
PMCID: PMC1828907  PMID: 17046718
PMAT; hENT4; serotonin; hybrid depletion; human brain; SSRIs
22.  Development and characterization of a recombinant madin-darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1) 
AAPS PharmSci  2004;6(1):77-85.
Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells. The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors. The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated. The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport. Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs.
doi:10.1208/ps060108
PMCID: PMC2750943  PMID: 18465260
rat; MRP1; drug resistance; chemotherapeutic agents; cytotoxicity; transport; ATP-binding cassette; transwell

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