Transporter Binding Sites Research Articles

Substrate and Inhibitor Binding to a Glutamate Transporter Homologue

Glutamate transporters tightly control extracellular glutamate concentration by pumping the transmitter into neurons and glia. Active transport is achieved by coupling the energy of the transmembrane ionic gradients to the conformational changes of the transporter, which alternates between outward- and inward-facing states with the substrate-binding site accessible from the extra- and intracellular solutions, respectively. In order to concentrate the substrate into the cytoplasm, the transporter's binding site must exhibit a high affinity from the outside and a low affinity from the inside. To better understand this change, we have characterized the binding properties of a prokaryotic homologue of the glutamate transporters, GltPh, by means of isothermal titration calorimetry. We designed double cysteine mutants that, upon cross-linking, lock the transporter in either the outward- or the inward-facing state and measured the thermodynamic binding parameters of the substrate aspartate and a competitive inhibitor TBOA. Surprisingly, the free energies of aspartate binding to the outward- and inward-facing states of the transporter are very similar at 25 °C, although the enthalpic and entropic contributions differ significantly. GltPh affinity for aspartate depends steeply on the concentration of sodium ions (Na), consistent with binding of ∼3 Na being thermodynamically coupled to binding of each substrate molecule. In contrast to the substrate, TBOA shows significantly lower affinity for the inward-facing state compared to the outward-facing state. In both states, TBOA exhibits a weaker sodium-dependence with an apparent number of coupled Na near 1. Our results show that in the absence of a sodium gradient, GltPh binds aspartate and Na with the same affinity in the outward- and inward-facing states. Therefore, the higher substrate affinity on the extracellular side of the membrane is determined by the higher Na concentration in the extracellular space compared to the cytoplasm.

Acidification increases mercury uptake by a freshwater alga, Chlamydomonas reinhardtii

Environmental contextMercury is classified as a priority pollutant owing to the biomagnification of its methylated species along food chains and the consequent effects on top consumers. The pH of natural waters affects many of the biogeochemical processes that control mercury accumulation in aquatic organisms. Here, evidence is presented that pH affects mercury uptake by unicellular algae, primary producers in aquatic food chains, thereby providing a new example of the pervasive influence of pH on the mercury biogeochemical cycle. AbstractWe have examined the influence of pH on HgII uptake (mainly in the form of the lipophilic complex HgCl2) by a green, unicellular alga, Chlamydomonas reinhardtii. Uptake of the dichloro complex increased by a factor of 1.6 to 2 when the pH was lowered from 6.5 to 5.5, an unexpected result given that the intracellular hydrolysis rate of fluorescein diacetate (FDA), used as a probe for the passive diffusion of lipophilic solutes through algal membranes, decreased in the studied alga under similar conditions. Several mechanisms were explored to explain the enhanced uptake at pH 5.5, including pH-induced changes in cell surface binding of Hg or in Hg loss rates from cells, but none of them gave completely satisfactory explanations. The present findings imply that inorganic HgII in aqueous solution behaves, in terms of uptake, neither as a lipophilic complex (the uptake of which would be expected to decrease with acidification because of algal membrane packing), nor as a cationic metal (the transport of which by facilitated transport would be expected to diminish with increasing proton concentration because of metal–proton competition at the transporter binding sites). Mercury uptake by algae seems rather to be stimulated by proton addition.

Pharmacological challenge and synaptic response – assessing dopaminergic function in the rat striatum with small animal single-photon emission computed tomography (SPECT) and positron emission tomography (PET)

Disturbances of dopaminergic neurotransmission may be caused by changes in concentrations of synaptic dopamine (DA) and/or availabilities of pre- and post-synaptic transporter and receptor binding sites. We present a series of experiments which focus on the regulatory mechanisms of the dopamin(DA)ergic synapse in the rat striatum. In these studies, DA transporter (DAT) and/or D(2) receptor binding were assessed with either small animal single-photon emission computed tomography (SPECT) or positron emission tomography (PET) after pharmacological challenge with haloperidol, L-DOPA and methylphenidate, and after nigrostriatal 6-hydroxydopamine lesion. Investigations of DAT binding were performed with [(123)I]N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl)nortropane ([(123)I]FP-CIT). D(2) receptor bindingd was assessed with either [(123)I](S)-2-hydroxy-3-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide ([(123)I]IBZM) or [(18)F]1[3-(4'fluorobenzoyl)propyl]-4-(2-keto-3-methyl-1-benzimidazolinyl)piperidine ([(18)F]FMB). Findings demonstrate that in vivo investigations of transporter and/or receptor binding are feasible with small animal SPECT and PET. Therefore, tracers that are radiolabeled with isotopes of comparatively long half-lives such as (123)I may be employed. Our approach to quantify DAT and/or D(2) receptor binding at baseline and after pharmacological interventions inducing DAT blockade, D(2) receptor blockade, and increases or decreases of endogenous DA concentrations holds promise for the in vivo assessment of synaptic function. This pertains to animal models of diseases associated with pre- or postsynaptic DAergic deficiencies such as Parkinson's disease, Huntington's disease, attention-deficit/hyperactivity disorder, schizophrenia or drug abuse.

Analysis of the bioavailability of Cr(III) and Cr(VI) based on the determination of chromium in Mentha piperita by graphite furnace atomic absorption spectrometry

Mentha piperita L. (Lamiace) was cultivated under the controlled laboratory conditions in the presence of varying levels of trivalent and hexavalent chromium in order to determine its capacity to control chromium uptake and its tolerance limit. The plants were grown in pots at 25?C with controlled soil moisture (about 80 % of the water retention capacity). The soil was treated with increasing concentrations of Cr(NO3)3 (40, 80, 120, and 200 mg kg-1) and K2Cr2O7 (2.5, 5, 10, and 15 mg kg-1). A control group of plants was grown without the addition of chromium to the soil. For each concentration, three acidity levels were tested: natural, one pH unit below and one above the natural acidity of the soil (pH2 = 6, pH1 = 5 and pH3 = 7). The plant samples were digested according to the standard procedure and chromium content was determined by GFAAS. For all plants, the transportation index was calculated and the results (expressed in mg kg-1) at pH1, pH2 and pH3, respectively, were: 0.21-0.80, 0.06-1.06 and 0.04-0.52. The recoveries were good (72.73-115.3 %) as evidenced by the analysis of certified reference materials (NIST SRM 8433 - Corn Bran and NIST SRM 1547 - Peach Leaves). The mobility of chromium through the plants tissues is discussed in regard to its competition with iron and manganese for transport binding sites; hence Mn and Fe were also determined.

Restorative Effects of Platelet Derived Growth Factor-BB in Rodent Models of Parkinson's Disease

Parkinson's disease is characterized by motor deficits caused by loss of midbrain dopaminergic neurons. Neurotrophic factors and cell transplantation have partially restored function in models of Parkinson's disease, but have had limited effects in humans. Here we show that intracerebroventricular administration of platelet-derived growth factor-BB can offer an alternative strategy to restore function in Parkinson's disease; In animal models of nigrostriatal injury, a two weeks treatment with platelet-derived growth factor-BB resulted in long-lasting restoration of striatal dopamine transporter binding sites and expression of nigral tyrosine hydroxylase. It also normalized amphetamine-induced rotational behavior in 6-hydroxydopamine lesioned rats. Platelet-derived growth factor-BB promoted proliferation of neural progenitor cells in the subventricular zone. The effects on dopaminergic neurons and functional recovery could be blocked by co-infusion with a proliferation inhibitor, indicating a link between the proliferative and anti-parkinsonian effects. Based on the current data, we consider platelet-derived growth factor-BB a clinical candidate drug for treatment of Parkinson's disease.

Differential response of the central noradrenergic nervous system to the loss of locus coeruleus neurons in Parkinson's disease and Alzheimer's disease

In Parkinson's disease (PD), there is a significant loss of noradrenergic neurons in the locus coeruleus (LC) in addition to the loss of dopaminergic neurons in the substantia nigra (SN). The goal of this study was to determine if the surviving LC noradrenergic neurons in PD demonstrate compensatory changes in response to the neuronal loss, as observed in Alzheimer's disease (AD). Tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) mRNA expression in postmortem LC tissue of control and age-matched PD subjects demonstrated a significant reduction in the number of noradrenergic neurons in the LC of PD subjects. TH mRNA expression/neuron did not differ between control and PD subjects, but DBH mRNA expression/neuron was significantly elevated in PD subjects compared to control. This increase in DBH mRNA expression in PD subjects is not a response to neuronal loss because the amount of DBH mRNA expression/neuron in AD subjects was not significantly different from control. Norepinephrine transporter (NET) binding site concentration in the LC of PD subjects was significantly reduced over the cell body region as well as the peri-LC dendritic zone. In PD subjects, the loss of dendrites from surviving noradrenergic neurons was also apparent with TH-immunoreactivity (IR). This loss of LC dendritic innervation in PD subjects as measured by TH-IR was not due to LC neuronal loss because TH-IR in AD subjects was robust, despite a similar loss of LC neurons. These data suggest that there is a differential response of the noradrenergic nervous system in PD compared to AD in response to the loss of LC neurons.

ChemInform Abstract: Synthesis, Dopamine and Serotonin Transporter Binding Affinities of Novel Analogues of Meperidine.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Mechanism of Interaction of the Glutamate Transporter EAAC1 with K+

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na+ and one H+, and the outward movement of one K+ ion. Internal K+ is known to bind to the transporter after glutamate and Na+ are unloaded to the cytosol, subsequently initiating relocation of the transporter binding sites to complete the transport cycle. However, parameters of K+ interaction with EAAC1, such as affinity and voltage dependence, are currently unknown. Here, we determined the steady-state and pre-steady state kinetics of the interaction of K+ with its extracellular binding site, and the subsequent K+ transport step, by using transport current recording from EAAC1-transfected cells. Our results show that K+ binds to its extracellular binding site with high affinity (Km = 4.5 mM). K+ affinity is only weakly voltage dependent. However, transient transport currents were observed in response to steps of the transmembrane potential when K+ was the only cation present. These currents were capacitive in nature and the charge movement followed a Boltzmann-like voltage dependence. Together, these results suggest that the cation binding process senses little of the transmembrane electric field, but that a subsequent K+-induced reaction step, possibly the K+-dependent transporter relocation, is electrogenic. The rate constant of the voltage dependent step was 70 s−1. This result is consistent with previous data that suggested the K+-induced relocation to be the rate-limiting step in the transport cycle. We propose a kinetic model, which is based on an alternating access mechanism, including a fast, voltage-independent K+ binding step and a slow, electrogenic conformational change. Our model can be used to predict the kinetics of the K+-dependent half-cycle of the glutamate transport process.This work was supported by NIH grant 2R01NS049335-06A1.

Homes for the orphans: utilization of multiple substrate-binding proteins by ABC transporters

Acquiring nutrients from the environment is essential for all microbes, and the ATP-binding cassette (ABC) transporters are one of the major routes by which bacteria achieve it. In this issue of Molecular Microbiology, Chen et al. describe their characterization of what appeared at first glance a simple ABC transporter for acquisition of quaternary ammonium compounds (QACs) in Pseudomonas sp., but their persistence in fully determining the properties of this system led to the experimental demonstration that QAC uptake utilizes three different substrate-binding proteins (SBPs), two of which are encoded at remote locations on the genome as 'orphan' SBPs that are each able to function with a single core ABC transporter. Building on the unusual nature of this system, in which multiple SBPs with non-overlapping substrate specificities compete for the same transporter binding site, they designed elegant in vivo experiments that suggest that only substrate-bound SBPs are able to form functional complexes with the membrane domains. This new finding provides an important piece of in vivo data leading to further insight into how this ubiquitous family of transporters operates.

Inverse relationship between numbers of 5-HT transporter binding sites and life history of aggression and intermittent explosive disorder

The objective of this study was to determine if platelet 5-HT transporter (5-HTT) sites vary as a function of aggression, and/or impulsiveness, and differ as a function of Intermittent Explosive Disorder (IED). Accordingly, the number of platelet 5-HTT sites was assessed in 100 personality disordered (PD) individuals with varying degrees of aggressiveness. The number of platelet 5-HTT sites was assessed by examining the Bmax of H 3-Paroxetine Binding to the blood platelet. Life history of aggression was assessed by Life History of Aggression. Impulsivity was assessed by the Barratt Impulsiveness Scale. Diagnoses of IED were made by both DSM-IV and Research Criteria. Examination of the data revealed that Bmax, but not Kd, values of Platelet H 3-Paroxetine Binding correlated inversely with the LHA Aggression score ( r = −.42 n = 87, p < .001) but not with the BIS-11 Impulsivity score ( r = .03, n = 77, p = .777). PD subjects meeting Research Criteria for IED demonstrated a significant reduction in Bmax values for Platelet H 3-Paroxetine Binding. These results were similar after accounting for the effect of lifetime history of depressive mood disorder on Bmax values for Platelet H 3-Paroxetine Binding. These data indicate a significant inverse relationship between platelet 5-HTT and aggression, though not impulsivity, as a dimensional variable in personality disordered individuals. Results from the examination of IED as a categorical aggression variable suggest that Research, rather than DSM-IV, criteria better identify individuals with reduced numbers of platelet 5-HTT sites.

Lack of CB1 receptor activity impairs serotonergic negative feedback

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behaviour and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB(1) cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB(1) knockout compared with wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB(1) knockout mice. The lack of CB(1) receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus (DRN) revealed that the genetic and pharmacological blockade of CB(1) receptor induced a 5-HT(1A) autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT(2C) receptor within several brain areas related to the control of the emotional responses, such as the DRN, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an over-expression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB(1) receptor induces a facilitation of the activity of serotonergic neurons in the DRN by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice.

In vitro binding assays using 3H nisoxetine and 3H WIN 35,428 reveal selective effects of gonadectomy and hormone replacement in adult male rats on norepinephrine but not dopamine transporter sites in the cerebral cortex

The prefrontal cortices mediate cognitive functions that critically depend on local dopamine levels. In male rats, many prefrontal tasks where performance is disrupted by changes in dopamine signaling are also impaired by gonadectomy, a manipulation that increases cortical dopamine concentration, prefrontal dopamine axon density and possibly extracellular prefrontal dopamine levels as well. Because these actions could be responsible for the impairing effects of gonadectomy on prefrontal function, the question of how they might arise comes to the fore. Accordingly, the present studies asked whether dopamine levels might be increased via a hormone sensitivity of transporter-mediated dopamine uptake. Specifically, 3H WIN 35,428 and 3H nisoxetine, ligands selective for the dopamine (DAT)- and norepinephrine transporter (NET) respectively, were used in in vitro binding assays to ask whether gonadectomy altered transporter affinity (Kd) and/or binding site number (Bmax) in prefrontal cortex, sensorimotor cortex and/or caudate. Assays performed on tissues dissected from sham-operated, gonadectomized and gonadectomized rats supplemented with testosterone propionate or estradiol for 4 or 28 days revealed no significant group differences or obvious trends in Kd or Bmax for DAT binding or in measures of Bmax for NET binding. However, affinity constants for 3H nisoxetine were found to be significantly higher in sensorimotor and/or prefrontal cortex of rats gonadectomized and gonadectomized and supplemented with estradiol for 4 or 28 days but similar to control in gonadectomized rats given testosterone. Because the NET contributes substantially to extracellular prefrontal dopamine clearance, these androgen-mediated effects could influence prefrontal dopamine levels and might thus be relevant for observed effects of gonadectomy on dopamine-dependent prefrontal behaviors. A hormone sensitivity of the NET could also have bearing on the prefrontal dopamine dysfunction seen in disorders like schizophrenia that disproportionately affect males, whose severity correlates with abnormal testosterone levels, and for which the NET is among suspected sites of pathology.

Long-term alterations in vulnerability to addiction to drugs of abuse and in brain gene expression after early life ethanol exposure

Exposure to ethanol early in life can have long-lasting implications on brain function and drug of abuse response later in life. The present study investigated in rats, the long-term consequences of pre- and postnatal (early life) ethanol exposure on drug consumption/reward and the molecular targets potentially associated with these behavioral alterations. Since a relationship has been demonstrated between heightened drugs intake and susceptibility to drugs-induced locomotor activity/sensitization, anxiolysis, we tested these behavioral responses, depending on the drug, in control and early life ethanol-exposed animals. Our results show that progeny exposed to early life ethanol displayed increased consumption of ethanol solutions and increased sensitivity to cocaine rewarding effects assessed in the conditioned place preference test. Offspring exposed to ethanol were more sensitive to the anxiolytic effect of ethanol and the increased sensitivity could, at least in part, explain the alteration in the consumption of ethanol for its anxiolytic effects. In addition, the sensitivity to hypothermic effects of ethanol and ethanol metabolism were not altered by early life ethanol exposure. The sensitization to cocaine (20 mg/kg) and to amphetamine (1.2 mg/kg) was increased after early life ethanol exposure and, could partly explain, an increase in the rewarding properties of psychostimulants. Gene expression analysis revealed that expression of a large number of genes was altered in brain regions involved in the reinforcing effects of drugs of abuse. Dopaminergic receptors and transporter binding sites were also down-regulated in the striatum of ethanol-exposed offspring. Such long-term neurochemical alterations in transmitter systems and in the behavioral responses to ethanol and other drugs of abuse may confer an increased liability for addiction in exposed offspring.

Ethanol potentiates dopamine uptake and increases cell surface distribution of dopamine transporters expressed in SK-N-SH and HEK-293 cells

Ethanol increases dopaminergic release in the reward and reinforcement areas of the brain. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT). In vitro electrophysiological and biochemical studies in Xenopus laevis oocytes have previously shown ethanol potentiates DAT function and increases transporter-binding sites. The potentiating effect of ethanol on the transporter is eliminated in Xenopus oocytes by the DAT mutation glycine 130 to threonine. However, ethanol's action on DAT functional regulation has yet to be examined in mammalian cell expression systems. To further understand the molecular mechanisms of ethanol's action on DAT, we determined the direct mechanistic action of short-term (≤2 h) ethanol exposure on transporter function and cell surface distribution in non-neuronal human embryonic kidney cells-293 (HEK-293) and neuronal SK-N-SH neuroblastoma cells expressing the transporter. Wild-type or G130T mutant DAT were overexpressed in HEK-293 and SK-N-SH cells. Ethanol potentiated DAT mediated [ 3H]DA uptake in a dose (25, 50, 100 mM), but not time dependent manner in cells expressing wild-type DAT. Ethanol-induced potentiation of uptake was significantly reduced in cells expressing the G130T mutant. Analysis of DA uptake kinetic parameters indicates 100-mM ethanol exposure increased [ 3H]DA uptake velocity ( V max), while affinity for DA ( K m) remained unchanged. The effect of ethanol on wild-type DAT surface expression was measured by biotinylation cell surface labeling. DAT surface expression increased 40%–50% after 1-h, 100-mM ethanol exposure. These studies show ethanol potentiates DAT functional regulation in both neuronal and non-neuronal cells, suggesting a direct mechanistic action of ethanol on transporter trafficking in mammalian systems. Our findings demonstrate ethanol's action on DAT function and regulation is consistent across multiple model systems.

EFFECTS OF INDOMETHACIN ON INTRACELLULAR CALCIUM AND PROTON REGULATORY MECHANISMS

Stilbenedisulfonates (SD) bind to a “primary” SD (PSD) site on the outer membrane surface of band 3, and inhibit anion exchange (AE) allosterically. Yet, evidence [Membr. Biochem. 2 (1979) 297] suggests that SD can be transported by band 3, thus raising questions about the relative locations of the transport and SD binding sites. A “second” class of DBDS (4,4′-dibenzamido-2,2′-stilbenedisulfonate) binding sites has been discovered, which is activated by protons (pK ∼ 5.0), and is located on the membrane domain of band 3 [Biochem. J. 388 (2005) 343]. Here we show that the “second” class of DBDS binding sites, not the PSD site, lies on the SD transport pathway. We compare the pH dependence of DBDS influx to DBDS binding using: (a) control cells, (b) cells selectively crosslinked at the PSD site by treatment with 300 μM BS3 (bis(sulfosuccinimidyl)suberate), and (c) cells with DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) bound covalently to the PSD site. DBDS binds to the “second” class of sites on band 3 in all three types of cells. DBDS does not bind to the PSD sites of BS3- or DIDS-modified cells. Proton-activated DBDS influx was observed using control and BS3-modified cells, but not when using DIDS-modified cells. The results with DIDS suggest that the PSD site and the transport site overlap. However, this interpretation is disproved by experiments with BS3-modified cells, where the PSD site is blocked, yet DBDS transport and binding to the “second” class of sites both take place.

3,4-Methylenedioxymethamphetamine induces differential regulation of tryptophan hydroxylase 2 protein and mRNA levels in the rat dorsal raphe nucleus

Previous investigations with 3,4-methylenedioxymethamphetamine (MDMA) have suggested that administration of this drug results in a degeneration of 5-HT nerve terminals and subsequent alterations in 5-HT neurotransmission. However, only limited investigations have examined the effects of MDMA on the dorsal raphe nucleus. The present study was designed to assess the effect of MDMA on the rate-limiting enzyme in 5-HT biosynthesis, tryptophan hydroxylase (TPH), by measuring TPH2 protein and mRNA levels in rat dorsal raphe (DR) nucleus. Rats were administered MDMA (20 mg/kg, s.c.) or saline twice daily for 4 days and killed 14 days later. Tissue sections of the DR were processed for quantitative immunoautoradiography and in situ hybridization histochemistry for measurements of the levels of TPH2-immunoreactivity (IR) and TPH2 mRNA. To assess 5-HT axon terminal integrity after MDMA treatment, the density of 5-HT transporter (SERT) binding sites was measured by quantitative autoradiography using [125I]RTI-55 ((-)-2beta-carbomethoxy-3 beta-(4-iodophenyl) tropane) ( 125I-RTI-55) as a ligand. TPH2-IR levels were significantly decreased by 45% in the mid DR and by 40% in the caudal DR in the MDMA-treated rats compared with saline-injected rats. In contrast, TPH2 mRNA levels were significantly increased by 24% in the mid DR and by 12% in the caudal DR. MDMA treatment significantly decreased 125I-RTI-55 labeled SERT binding sites in the striatum, nucleus accumbens and cingulate cortex demonstrating a loss of 5-HT terminals. The increase in TPH2 mRNA levels in both the mid DR and caudal DR of MDMA-treated rats may reflect a compensatory mechanism in the injured 5-HT neurons to increase TPH2 protein synthesis. Taken together, our results suggest that a serious defect occurs in the biosynthesis of TPH2 in the DR following MDMA administration.

Cocaine Seeking Habits Depend upon Dopamine-Dependent Serial Connectivity Linking the Ventral with the Dorsal Striatum

A neuroanatomical principle of striatal organization has been established through which ventral domains, including the nucleus accumbens, exert control over dorsal striatal processes mediated by so-called "spiraling," striato-nigro-striatal, circuitry. We have investigated the functional significance of this circuitry in the control over a cocaine-seeking habit by using an intrastriatal disconnection procedure that combined a selective, unilateral lesion of the nucleus accumbens core and infusion of a dopamine receptor antagonist into the contralateral dorsolateral striatum, thereby disrupting striato-midbrain-striatal serial connectivity bilaterally. We show that this disconnection selectively decreased drug-seeking behavior in rats extensively trained under a second-order schedule of cocaine reinforcement. These data thereby define the importance of interactions between ventral and dorsal domains of the striatum, mediated by dopaminergic transmission, in the neural mechanisms underlying the development and performance of cocaine-seeking habits that are a key characteristic of drug addiction.

MDMA modifies active avoidance learning and recall in mice

Several studies have suggested the existence of cognitive deficits after repeated or high doses of 3,4-methylenedioxymethamphetamine (MDMA) in humans and experimental animals. However, the extent of the impairments observed in learning or memory tasks remains unclear. The objective of this study was to evaluate the effects of different dosing regimens of MDMA on the ability of mice to learn and recall an active avoidance task. Animals were treated with MDMA (0, 1, 3, 10 and 30 mg/kg) under four different experimental conditions, and active avoidance acquisition and recall were evaluated. In experiments 1 and 2, MDMA was administered 1 h before different active avoidance training sessions. In experiments 3 and 4, mice received a repeated treatment with MDMA before or after active avoidance training, respectively. Changes in presynaptic striatal dopamine transporter (DAT) binding sites were evaluated at two different time points in animals receiving a high dose of MDMA (30 mg/kg) or saline twice a day over 4 days. MDMA administered before the active avoidance sessions interfered with the acquisition and the execution of a previously learned task. A repeated treatment with high doses of MDMA administered before training reduced acquisition of active avoidance in mice, while pre-treatment with both high and low doses of MDMA impaired recall of this task. A reduction in DAT binding was observed 4 days but not 23 days after the last MDMA administration. Acute MDMA modifies the acquisition and execution of active avoidance in mice, while repeated pre-treatment with MDMA impairs acquisition and recall of this task.

Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.

Structural Variation Governs Substrate Specificity for Organic Anion Transporter (OAT) Homologs: POTENTIAL REMOTE SENSING BY OAT FAMILY MEMBERS

Organic anion transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over 40 structurally diverse organic anions, we find a correlation between organic anion potency (pKi) and hydrophobicity (logP) suggesting a hydrophobicity-driven association with transporter-binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative molecular field analysis confirms these mechanistic insights and provides a model for predicting new OAT substrates. By comparative molecular field analysis, both hydrophobic and charged interactions contribute to Oat1 binding, although it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the three-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (Vmax) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity for a particular transporter may enable design of drugs for targeting to specific tissues (e.g. olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g. kidney, olfactory mucosa) via organic anions.