[Show abstract][Hide abstract] ABSTRACT: Recent aggregation of evidence for the roles of endogenous agonist and receptor systems that are mimicked or activated by cannabanoid ligands has provided a focus for work that has elucidated details of some of the multiple physiological roles and pharmacological functions that these systems play in brain and peripheral tissues. This chapter reviews some of the approaches to improved elucidation of these systems, with special focus on the human genes that encode cannabanoid receptors and the variants in these receptors that appear likely to contribute to human addiction vulnerabilities.
[Show abstract][Hide abstract] ABSTRACT: 1. Individuals display significant differences in their levels of expression of the dopamine transporter (DAT; SLC6A3). These differences in DAT are strong candidates to contribute to individual differences in motor, mnemonic and reward functions. To identify "cis"-acting genetic mechanisms for these individual differences, we have sought variants in 5' aspects of the human DAT gene and identified the haplotypes that these variants define. 2. We report (i) significant relationships between 5' DAT haplotypes and human individual differences in ventral striatal DAT expression assessed in vivo using [(11)C] cocaine PET and (ii) apparent confirmation of these results in studies of DAT expression in postmortem striatum using [(3)H] carboxyflurotropane binding. 3. These observations support the idea that cis-acting variation in 5' aspects of the human DAT/SLC6A3 locus contributes to individual differences in levels of DAT expression in vivo. 5' DAT variation is thus a good candidate to contribute to individual differences in a number of human phenotypes.
Cellular and Molecular Neurobiology 01/2006; 26(4-6):875-89. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ala and Gly substitutions for Pro 101 (P101) located in transmembrane domain 2 of the dopamine transporter (DAT) abolished transport activity but did not disrupt plasma membrane expression. Due to the high conservation of P101 in all neurotransmitter transporters and the capability of Pro to add flexibility to helices, we hypothesized that P101 contributes to the dynamic feature of substrate translocation. To test this hypothesis, here we analysed transport activity for DAT mutants where this Pro was mutated into different amino acids, including Ser, Val, Leu and Phe. The transmembrane domain 2 helix of P101F, unlike the other mutants, was computationally predicted to have a Van der Waals energy threefold higher than the wild-type helix. P101F mutant expression was consistently disrupted in COS cells. Among all the other mutants that express normally, P101V, with a side-chain size close to that of Pro, restores the transport activity of P101A by sevenfold. Most importantly, P101V, P101L and P101S display negative-dosage effects on dopamine (DA) transport, i.e. the velocity-concentration curve for DA uptake does not show a plateau with increasing [DA] but rather peaks and then goes down. These data support the view that P101 of DAT plays an essential role in DA translocation.
Journal of Neurochemistry 08/2005; 94(1):276-87. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The vesicular monoamine transporter 2 (VMAT2, SLC18A2) takes up cytosolic monoamines into intracellular secretory vesicles, preventing their neurotoxicity in the cytosol and discharging them into extracellular space by exocytosis. It has been shown that one-copy deletion of the VMAT2 gene increases locomotion activity significantly in response to drug treatments and dopamine neuron death rate in response to neurotoxin treatments in knockout mice. Little is known about promoter polymorphisms and their influence on SLC18A2 promoter activity. We have re-sequenced a 17.4 kb DNA in the SLC18A2 promoter region for Caucasians and revealed 47 polymorphisms that confer 13 haplotypes. One of the haplotypes reaches a frequency as high as 65%, likely due to positive selection. In vitro analysis showed a 20% difference in promoter activity between two frequent haplotypes and identified some of the polymorphisms that influence promoter activity. Four haplotype-defining single nucleotide polymorphisms (hdSNPs) can define the frequent haplotypes and by genotyping these hdSNPs, we find that haplotypes with -14234G and -2504C of SLC18A2 promoter region represent a protective factor against alcoholism (P = 0.0038 by Fisher's exact tests). Therefore, SLC18A2 promoter haplotypes defined here create a foundation for transcriptional characterization of individuality and for association study on monoamine-related human diseases.
Human Molecular Genetics 06/2005; 14(10):1393-404. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 5-Hydroxytryptamine (serotonin) receptor 2B (HTR2B) is involved in brain development. Although expressed in the human brain, HTR2B has not been investigated much for its role in higher brain functions. Here we describe a genome-scan with 391 simple sequence repeat markers in 300 Caucasians, identifying HTR2B gene as a candidate for drug abuse vulnerability.
From DNA re-sequencing of 110 subjects, we discovered three novel single nucleotide polymorphisms (SNPs), two of which confer a double-mutant of the receptor protein in a drug-abusing population. Arg6, a conserved basic residue, and the conserved acidic Glu42 are mutated simultaneously into Gly, termed R6G/E42G. Furthermore, this double-mutant tends to associate with drug abuse (P = 0.08 by chi2 test). The third SNP that is a synonymous mutation in the codon of Gln11 showed significant association with drug abuse (P = 0.0335 by Fisher's exact test).
Our data are the first suggesting that HTR2B contributes to brain architecture and pathways that are involved in illegal drug reward.
[Show abstract][Hide abstract] ABSTRACT: Following exocytotic release of the biogenic amine neurotransmitters, norepinephrine and dopamine, are removed from the synaptic cleft by the respective transporter, norepinephrine transporter (NET) and dopamine transporter (DAT) located on the plasma membrane. The catecholamine transporters are the molecular targets for psychoactive drugs as well as drugs of abuse such as cocaine and amphetamine and the Parkinsonism-inducing neurotoxin, MPP+. Nicotine regulates the transport of catecholamines and MPP+ and may exert self-medicating effects for depression, schizophrenia and attention deficit hyperactivity disorder, and neuroprotective effects against MPP+ through the regulation of the transporters. The availability of cDNAs of these transporters has permitted detailed pharmacological studies in heterologous expression systems for determining the mechanisms of action of nicotine on transporters. Moreover, functional analysis of the effect of single amino acid substitution suggests that specific residues in DAT molecules may play a significant role in interaction with MPP+ and cocaine, and thus would promise a development of novel drugs with diverse chemical structures.
Nihon shinkei seishin yakurigaku zasshi = Japanese journal of psychopharmacology 05/2004; 24(2):43-7.
[Show abstract][Hide abstract] ABSTRACT: The activities of PP1 (protein phosphatase 1), a principal cellular phosphatase that reverses serine/threonine protein phosphorylation, can be altered by inhibitors whose activities are themselves regulated by phosphorylation. We now describe a novel PKC (protein kinase C)-dependent PP1 inhibitor, namely GBPI (gut and brain phosphatase inhibitor). The shorter mRNA that encodes this protein, GBPI-1, is expressed in brain, stomach, small intestine, colon and kidney, whereas a longer GBPI-2 splice variant mRNA is found in testis. Human GBPI-1 mRNA encodes a 145-amino-acid, 16.5 kDa protein with pI 7.92. GBPI contains a consensus PP1-binding motif at residues 21-25 and consensus sites for phosphorylation by enzymes, including PKC, PKA (protein kinase A or cAMP-dependent protein kinase) and casein kinase II. Recombinant GBPI-1-fusion protein inhibits PP1 activity with IC50=3 nM after phosphorylation by PKC. Phospho-GBPI can even enhance PP2A activity by >50% at submicromolar concentrations. Non-phosphorylated GBPI-1 is inactive in both assays. Each of the mutations in amino acids located in potential PP1-binding sequences, K21E+K22E and W25A, decrease the ability of GBPI-1 to inhibit PP1. Mutations in the potential PKC phosphoacceptor site T58E also dramatically decrease the ability of GBPI-1 to inhibit PP1. Interestingly, when PKC-phosphorylated GBPI-1 is further phosphorylated by PKA, it no longer inhibits PP1. Thus, GBPI-1 is well positioned to integrate PKC and PKA modulation of PP1 to regulate differentially protein phosphorylation patterns in brain and gut. GBPI, its closest family member CPI (PKC-potentiated PP1 inhibitor) and two other family members, kinase-enhanced phosphatase inhibitor and phosphatase holoenzyme inhibitor, probably modulate integrated control of protein phosphorylation states in these and other tissues.
[Show abstract][Hide abstract] ABSTRACT: Our laboratory and others elucidated the primary amino acid sequences of the dopamine transporter (DAT) by cloning its cDNA and genomic sequences more than 12 years ago. Motivations for this work included the ideas that cocaine's interactions with DAT accounted for its rewarding properties and that selective inhibitors of DAT/cocaine interactions might thus provide good anticocaine medications. Such ideas supported interest in the detailed structure-function relationships of cocaine/DAT interactions, and in the construction and characterization of extensive series of site-directed DAT mutants. We can now select the most interesting 20 cocaine-analog selective mutations of the more than 100 single- and multiple amino acid substitution mutations that we have characterized. These mutants selectively reduce the affinities of the mutant DATs for cocaine analogs, but (absolutely or relatively) spare their affinities for dopamine. Several themes relevant to cocaine/DAT interactions emerge from these mutants. First, such mutations are found in a number of different DAT domains. Secondly, many but not all of these mutations lie in groups, near each other and near the same faces of presumably helical DAT transmembrane domains. Third, most are also conserved in the serotonin transporter (SERT), a transporter that is now strongly implicated in cocaine reward based on data from knockout mice. We discuss the results from these "top 20" mutants in light of the strengths and limitations of current DAT models and data from other studies. Taken together, these studies appear to indicate direct or indirect participation of several specific portions of DAT in selective recognition of cocaine analogs. These studies provide a strong basis for redirected studies aimed at producing dopamine- and serotonin-sparing cocaine antagonists that would represent combined DAT/SERT disinhibitors.
European Journal of Pharmacology 11/2003; 479(1-3):71-82. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca2+/calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake Vmax. PKC principally modulates Vmax. Neither p38 nor Ca2+/calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. 32P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.
Journal of Biological Chemistry 06/2003; 278(22):20162-70. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human dopamine transporter (DAT, SLC6A3) is an important 15 exon gene for dopamine neurotransmission and dopamine recycling. Common exon 15 variable number tandem repeat variants can be associated with attention deficit/hyperactivity disorder. Rarer single nucleotide polymorphisms produce missense variants including V55A and V382A. We now report studies of the functional influences of these DAT protein-coding variants. In COS cell transient-expression assays, V382A displays about half of the dopamine uptake velocity Vmax and cocaine analog binding Bmax values of wildtype DAT. V382A lowers dopamine's potency in inhibiting cocaine analog binding by six-fold. Cells expressing V382A or mixtures of V382A and wildtype DAT both display reduced plasma membrane and increased perinuclear expression, consistent with dominant effects of V328A on expression. V55A expresses normally but reveals a 1.7-fold-lower Km for dopamine uptake. Individuals with these human DAT protein variants could display altered dopamine systems.
The Pharmacogenomics Journal 02/2003; 3(3):159-68. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dopamine transporter (DAT) can mediate not only inward uptake of dopamine, but also its outward efflux by mechanisms that have been only partially elucidated. DAT-dependent dopamine efflux can be studied kinetically and apparent substrate affinity and V(max) values determined. We now report that wild-type DAT displays apparent affinities for efflux more than 300-fold lower than those for uptake. Efflux rates are enhanced by increased extracellular concentrations of dopamine or amphetamine and by lowered extracellular concentrations of Na(+) or Cl(-). Alanine substitutions for six proline residues located in or near DAT transmembrane domains increase apparent affinity and decrease V(max) values for dopamine efflux mediated by these mutant transporters. Mutant 12P572A displays increased DAT efflux with reduced dependence on ion or dopamine concentrations. These data add to evidence for the specificity of transporter-mediated efflux processes and begin to elucidate DAT candidate domains that may be preferentially involved with efflux activities.
Molecular Brain Research 01/2003; 108(1-2):71-80. · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cocaine's blockade of dopamine reuptake by brain dopamine transporters (DAT) is a central feature of current understanding of cocaine reward and addiction. Empirical screening of small-molecule chemical libraries has thus far failed to provide successful cocaine blockers that allow dopamine reuptake in the presence of cocaine and provide cocaine "antagonism". We have approached this problem by assessing expression, dopamine uptake, and cocaine analog affinities of 56 DAT mutants in residues located in or near transmembrane domains likely to play significant roles in cocaine recognition and dopamine uptake. A phenylalanine-to-alanine mutant in putative DAT transmembrane domain 3, F154A, retains normal dopamine uptake, lowers cocaine affinity 10-fold, and reduces cocaine stereospecificity. Such mutants provide windows into DAT structures that could serve as targets for selective cocaine blockers and document how combined strategies of mutagenesis and small molecule screening may improve our abilities to identify and design compounds with such selective properties.
[Show abstract][Hide abstract] ABSTRACT: This review presents the remarkable advances that have been achieved in marijuana (cannabinoid) research, with the discovery of specific receptors and the existence of naturally occurring cannabis-like substances in the human body and brain. The last decade has seen more rapid progress in marijuana research than any time in the thousands of years that marijuana has been used by humans, particularly in cannabinoid genomics. The cDNA and genomic sequences encoding G protein-coupled cannabinoid receptors (Cnrs) from several species have now been cloned. Endogenous cannabinoids (endocannabinoids), synthetic and hydrolyzing enzymes and transporters that define neurochemically-specific cannabinoid brain pathways have been identified. Endocannabinoid lipid signaling molecules alter activity at G protein-coupled receptors (GPCR) and possibly at anandamide-gated ion channels, such as vanilloid receptors. Availability of increasingly-specific CB1 and CB2 Cnr antagonists and of CB1 and CB2 Cnr knockout mice have increased our understanding of these cannabinoid systems and provides tantalizing evidence for even more G protein-coupled Cnrs. Initial studies of the Cnr gene structure, regulation and polymorphisms whet our appetite for more information about these interesting genes, their variants and roles in vulnerabilities to addictions and other neuropsychiatric disorders. Behavioral studies of cannabinoids document the complex interactions between rewarding and aversive effects of these drugs. Pursuing cannabinoid-related molecular, pharmacological and behavioral leads will add greatly to our understanding of endogenous brain neuromodulator systems, abused substances and potential therapeutics. This review of CB1 and CB2 Cnr genes in human and animal brain and their neurobiological effects provide a basis for many of these studies. Therefore, understanding the physiological cannabinoid control system in the human body and brain will contribute to elucidating this natural regulatory mechanism in health and disease.
Progress in Neurobiology 05/2002; 66(5):307-44. · 9.04 Impact Factor