Howard H. Gu

Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs

BackgroundThe plasma membrane neurotransmitter transporters terminate neurotransmissions by the reuptake of the released neurotransmitters. The transporters for the monoamines dopamine, norepinephrine, and serotonin (DAT, NET, and SERT) are targets for several popular psychostimulant drugs of abuse. The potencies of the psychostimulant on the monoamine transporters have been studied by several laboratories. However, there are significant discrepancies in the reported data with differences up to 60-fold. In addition, the drug potencies of the 3 monoamine transporters from mouse have not been compared in the same experiments or along side the human transporters. Further studies and systematic comparisons are needed.ResultsIn this study, we compared the potencies of five psychostimulant drugs to inhibit human and mouse DAT, SERT and NET in the same cellular background. The KI values of cocaine to inhibit the 3 transporters are within a narrow range of 0.2 to 0.7 μM. In comparison, methylphenidate inhibited DAT and NET at around 0.1 μM, while it inhibited SERT at around 100 μM. The order of amphetamine potencies was NET (KI = 0.07–0.1 μM), DAT (KI ≈ 0.6 μM), and SERT (KI between 20 to 40 μM). The results for methamphetamine were similar to those for amphetamine. In contrast, another amphetamine derivative, MDMA (3–4 methylenedioxymethamphetamine), exhibited higher potency at SERT than at DAT. The human and mouse transporters were similar in their sensitivities to each of the tested drugs (KI values are within 4-fold).ConclusionThe current and previous studies support the following conclusions: 1) cocaine blocks all 3 monoamine transporters at similar concentrations; 2) methylphenidate inhibits DAT and NET well but a 1000-fold higher concentration of the drug is required to inhibit SERT; 3) Amphetamine and methamphetamine are most potent at NET, while being 5- to 9-fold less potent at DAT, and 200- to 500-fold less potent at SERT; 4) MDMA has moderately higher apparent affinity for SERT and NET than for DAT. The relative potencies of a drug to inhibit DAT, NET and SERT suggest which neurotransmitter systems are disrupted the most by each of these stimulants and thus the likely primary mechanism of drug action.

Cell-specific Sorting of Biogenic Amine Transporters Expressed in Epithelial Cells

We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT). These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques. Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes. Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results. Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side. Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results. These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.

Dopamine Transporter Gene Variant Affecting Expression in Human Brain is Associated with Bipolar Disorder

The gene encoding the dopamine transporter (DAT) has been implicated in CNS disorders, but the responsible polymorphisms remain uncertain. To search for regulatory polymorphisms, we measured allelic DAT mRNA expression in substantia nigra of human autopsy brain tissues, using two marker SNPs (rs6347 in exon 9 and rs27072 in the 3′-UTR). Allelic mRNA expression imbalance (AEI), an indicator of cis-acting regulatory polymorphisms, was observed in all tissues heterozygous for either of the two marker SNPs. SNP scanning of the DAT locus with AEI ratios as the phenotype, followed by in vitro molecular genetics studies, demonstrated that rs27072 C>T affects mRNA expression and translation. Expression of the minor T allele was dynamically regulated in transfected cell cultures, possibly involving microRNA interactions. Both rs6347 and rs3836790 (intron8 5/6 VNTR) also seemed to affect DAT expression, but not the commonly tested 9/10 VNTR in the 3′UTR (rs28363170). All four polymorphisms (rs6347, intron8 5/6 VNTR, rs27072 and 3′UTR 9/10 VNTR) were genotyped in clinical cohorts, representing schizophrenia, bipolar disorder, depression, and controls. Only rs27072 was significantly associated with bipolar disorder (OR=2.1, p=0.03). This result was replicated in a second bipolar/control population (OR=1.65, p=0.01), supporting a critical role for DAT regulation in bipolar disorder.

Lack of Cocaine Self-Administration in Mice Expressing a Cocaine-Insensitive Dopamine Transporter

Cocaine addiction is a worldwide public health problem for which there are no established treatments. The dopamine transporter (DAT) is suspected as the primary target mediating cocaines abuse-related effects based on numerous pharmacological studies. However, in a previous study, DAT knockout mice were reported to self-administer cocaine, generating much debate regarding the importance of the DAT in cocaines abuse-related effects. Here, we show that mice expressing a “knockin” of a cocaine-insensitive but functional DAT did not self-administer cocaine intravenously despite normal food-maintained responding and normal intravenous self-administration of amphetamine and a direct dopamine agonist. Our results have three implications. First, they imply a crucial role for high-affinity DAT binding of cocaine in mediating its reinforcing effects, reconciling mouse genetic engineering approaches with data from classic pharmacological studies. Second, they demonstrate the usefulness of knockin strategies that modify specific amino acid sequences within a protein. Third, they show that it is possible to alter the DAT protein sequence in such a way as to selectively target its interaction with cocaine, while sparing other behaviors dependent on DAT function. Thus, molecular engineering technology could advance the development of highly specialized compounds such as a dopamine-sparing “cocaine antagonist.”

Mechanism for Cocaine Blocking the Transport of Dopamine: Insights from Molecular Modeling and Dynamics Simulations

Molecular modeling and dynamics simulations have been performed to study how cocaine inhibits dopamine transporter (DAT) for the transport of dopamine. The computationally determined DAT-ligand binding mode is totally different from the previously proposed overlap binding mode in which cocaine- and dopamine-binding sites are the same (Beuming, T.; et al. Nat. Neurosci. 2008, 11, 780-789). The new cocaine-binding site does not overlap with, but is close to, the dopamine-binding site. Analysis of all results reveals that when cocaine binds to DAT, the initial binding site is likely the one modeled in this study because this binding site can naturally accommodate cocaine. Then cocaine may move to the dopamine-binding site after DAT makes some necessary conformational change and expands the binding site cavity. It has been demonstrated that cocaine may inhibit the transport of dopamine through both blocking the initial DAT-dopamine binding and reducing the kinetic turnover of the transporter following the DAT-dopamine binding. The relative contributions to the phenomenological inhibition of the transport of dopamine from blocking the initial binding and reducing the kinetic turnover can be different in different types of assays. The obtained general structural and mechanistic insights are consistent with available experimental data and could be valuable for guiding future studies toward understanding cocaines inhibiting of other transporters.

Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression.

BackgroundThe dopamine transporter (DAT) plays a critical role in regulating dopamine neurotransmission. Variations in DAT or changes in basal dopaminergic tone have been shown to alter behavior and drug responses. DAT is one of the three known high affinity targets for cocaine, a powerful psychostimulant that produces reward and stimulates locomotor activity in humans and animals. We have shown that cocaine no longer produces reward in knock-in mice with a cocaine insensitive mutant DAT (DAT-CI), suggesting that cocaine inhibition of DAT is critical for its rewarding effect. However, in DAT-CI mice, the mutant DAT has significantly reduced uptake activity resulting in elevated basal dopaminergic tone, which might cause adaptive changes that alter responses to cocaine. Therefore, the objective of this study is to determine how elevated dopaminergic tone affects how mice respond to cocaine.ResultsWe examined the cocaine induced behavior of DAT knockdown mice that have DAT expression reduced by 90% when compared to the wild type mice. Despite a dramatic reduction of DAT expression and marked elevation in basal dopamine tone, cocaine produced reward, as measured by conditioned place preference, and stimulated locomotor activity in these mice.ConclusionA reduction in DAT expression and elevation of dopaminergic tone do not lead to adaptive changes that abolish the rewarding and stimulating effects of cocaine. Therefore, the lack of reward to cocaine observed in DAT-CI mice is unlikely to have resulted from the reduced DAT activity but instead is likely due to the inability of cocaine to block the mutated DAT and increase extracellular dopamine. This study supports the conclusion that the blockade of DAT is required for cocaine reward and locomotor stimulation.

A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate

Previously, we reported that Phe105 in transmembrane domain 2 of the mouse dopamine transporter (DAT) is crucial for high‐affinity cocaine binding. In the current study, we investigated whether other residues surrounding Phe105 also affect the potency of cocaine inhibition. After three rounds of sequential random mutagenesis at these residues, we found a triple mutant (L104V, F105C and A109V) of mouse DAT that retained over 50% uptake activity and was 69‐fold less sensitive to cocaine inhibition when compared with the wild‐type mouse DAT. The triple mutation also resulted in a 47‐fold decrease in sensitivity to methylphenidate inhibition, suggesting that the binding sites for cocaine and methylphenidate may overlap. In contrast, the inhibition of dopamine uptake by amphetamine or methamphetamine was not significantly changed by the mutations, suggesting that the binding sites for the amphetamines differ from those for cocaine and methylphenidate. Such functional but cocaine‐insensitive DAT mutants can be used to generate a knock‐in mouse line to study the role of DAT in cocaine addiction.

Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine

Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D2 receptor (D2R) function. However, although we demonstrated that striatal D1 receptor (D1R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D1Rs since haloperidol, by blocking the tonic inhibition of D2R, unmasked a normal activation of striatal adenosine A2A receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D1R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels.

Financial and Psychological Risk Attitudes Associated with Two Single Nucleotide Polymorphisms in the Nicotine Receptor (CHRNA4) Gene

With recent advances in understanding of the neuroscience of risk taking, attention is now turning to genetic factors that may contribute to individual heterogeneity in risk attitudes. In this paper we test for genetic associations with risk attitude measures derived from both the psychology and economics literature. To develop a long-term prospective study, we first evaluate both types of risk attitudes and find that the economic and psychological measures are poorly correlated, suggesting that different genetic factors may underlie human response to risk faced in different behavioral domains. We then examine polymorphisms in a spectrum of candidate genes that affect neurotransmitter systems influencing dopamine regulation or are thought to be associated with risk attitudes or impulsive disorders. Analysis of the genotyping data identified two single nucleotide polymorphisms (SNPs) in the gene encoding the alpha 4 nicotine receptor (CHRNA4, rs4603829 and rs4522666) that are significantly associated with harm avoidance, a risk attitude measurement drawn from the psychology literature. Novelty seeking, another risk attitude measure from the psychology literature, is associated with several COMT (catechol-O-methyl transferase) SNPs while economic risk attitude measures are associated with several VMAT2 (vesicular monoamine transporter) SNPs, but the significance of these associations did not withstand statistical adjustment for multiple testing and requires larger cohorts. These exploratory results provide a starting point for understanding the genetic basis of risk attitudes by considering the range of methods available for measuring risk attitudes and by searching beyond the traditional direct focus on dopamine and serotonin receptor and transporter genes.

Dopamine transporter inhibition is necessary for cocaine-induced increases in dendritic spine density in the nucleus accumbens.

Repeated exposure to cocaine produces changes in the nervous system that facilitate drug‐seeking behaviors. These drug‐seeking behaviors have been studied with animal models, such as cocaine‐induced locomotor sensitization. Cocaine is hypothesized to induce locomotor sensitization by neural changes, including an increase in the density of spines on the dendrites of neurons in the nucleus accumbens (NAC). However, how cocaine increases dendritic spine density in the NAC has been difficult to discern because cocaine inhibits the function of multiple targets, including the transporters for dopamine, serotonin, and norepinephrine. Previously, our lab created a tool that is useful for determining how inhibiting the dopamine transporter (DAT) contributes to the effects of cocaine by generating mice that express a cocaine‐insensitive DAT (DAT‐CI mice). In this study, we used DAT‐CI mice to determine the contribution of DAT inhibition in cocaine‐induced increases in dendritic spine density in the NAC. We repeatedly injected DAT‐CI mice with either cocaine or saline, and measured both dendritic spine density in the NAC and locomotor activity. Unlike wild‐type mice, DAT‐CI mice did not show an increase in dendritic spine density in the NAC or in locomotor activity in response to repeated injections of cocaine. These data show that cocaine‐induced increases in dendritic spine density in the NAC require DAT inhibition. Thus, DAT‐inhibition may play a role in mediating the long‐lasting neural changes associated with drug addiction. Synapse, 2011.