Elevated protease-activated receptor 4 (PAR4) gene expression in Alzheimer's disease predicts cognitive decline.

Platelet activation of protease-activated receptor 4 (PAR4) and thrombin are at the top of a chain of events leading to fibrin deposition, microinfarcts, blood-brain barrier disruption, and inflammation. We evaluated mRNA expression of the PAR4 gene F2RL3 in human brain and global cognitive performance in participants with and without cognitive impairment or dementia. Data were acquired from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP). F2RL3 mRNA was elevated in AD cases and was associated with worse retrospective longitudinal cognitive performance. Moreover, F2RL3 expression interacted with clinical AD diagnosis on longitudinal cognition whereas this relationship was attenuated in individuals without cognitive impairment. Additionally, when adjusting for the effects of AD neuropathology, F2RL3 expression remained a significant predictor of cognitive decline. F2RL3 expression correlated positively with transcript levels of proinflammatory markers including TNFα, IL-1ß, NFκB, and fibrinogen α/ß/γ. Together, these results reveal that F2RL3 mRNA expression is associated with multiple AD-relevant outcomes and its encoded product, PAR4, may play a role in disease pathogenesis.

Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening.

Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.

Role of protease-activated receptor 4 in mouse models of acute and chronic kidney injury.

Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor activated by thrombin. In the platelet, response to thrombin PAR4 contributes to the predominant procoagulant microparticle formation, increased fibrin deposition, and initiation of platelet-stimulated inflammation. In addition, PAR4 is expressed in other cell types, including endothelial cells. Under inflammatory conditions, PAR4 is overexpressed via epigenetic demethylation of the PAR4 gene, F2RL3. PAR4 knockout (KO) studies have determined a role for PAR4 in ischemia-reperfusion injury in the brain, and PAR4 KO mice display normal cardiac function but present less myocyte death and cardiac dysfunction in response to acute myocardial infarction. Although PAR4 has been reported to be expressed within the kidney, the contribution of PAR4 to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 KO mice are protected against kidney injury in two mouse models. First, PAR4 KO mice are protected against induction of markers of both fibrosis and inflammation in two different models of kidney injury: 1) 7 days following unilateral ureter obstruction (UUO) and 2) an AKI-CKD model of ischemia-reperfusion followed by 8 days of contralateral nephrectomy. We further show that PAR4 expression in the kidney is low in the control mouse kidney but induced over time following UUO. PAR4 KO mice are protected against blood urea nitrogen (BUN) and glomerular filtration rate (GFR) kidney function pathologies in the AKI-CKD model. Following the AKI-CKD model, PAR4 is expressed in the collecting duct colocalizing with Dolichos biflorus agglutinin (DBA), but not in the proximal tubule with Lotus tetragonolobus lectin (LTL). Collectively, the results reported in this study implicate PAR4 as contributing to the pathology in mouse models of acute and chronic kidney injury.NEW & NOTEWORTHY The contribution of the thrombin receptor protease-activated receptor 4 (PAR4) to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 expression is upregulated after kidney injury and PAR4 knockout (KO) mice are protected against fibrosis following kidney injury in two mouse models. First, PAR4 KO mice are protected against unilateral ureter obstruction. Second, PAR4 KO mice are protected against an AKI-CKD model of ischemia-reperfusion followed by contralateral nephrectomy.

Specificities of Gßγ subunits for the SNARE complex before and after stimulation of α2a-adrenergic receptors.

Ligand binding to G protein­coupled receptors (GPCRs), such as the α2a-adrenergic receptor (α2aAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gßγ dimers. α2aAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gßγ effector. To understand how the Gßγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gßγ subunits for the SNARE complex in adrenergic neurons, in which auto-α2aARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α2aARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gß and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gß1 and Gγ3 interacted with the SNARE complex. However, Gß1, Gß2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gßγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.

Identifying dominant-negative actions of a dopamine transporter variant in patients with parkinsonism and neuropsychiatric disease.

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

Using What We Already Have: Uncovering New Drug Repurposing Strategies in Existing Omics Data.

The promise of drug repurposing is to accelerate the translation of knowledge to treatment of human disease, bypassing common challenges associated with drug development to be more time- and cost-efficient. Repurposing has an increased chance of success due to the previous validation of drug safety and allows for the incorporation of omics. Hypothesis-generating omics processes inform drug repurposing decision-making methods on drug efficacy and toxicity. This review summarizes drug repurposing strategies and methodologies in the context of the following omics fields: genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, phenomics, pregomics, and personomics. While each omics field has specific strengths and limitations, incorporating omics into the drug repurposing landscape is integral to its success.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Bile diversion, a bariatric surgery, and bile acid signaling reduce central cocaine reward.

The gut-to-brain axis exhibits significant control over motivated behavior. However, mechanisms supporting this communication are poorly understood. We reveal that a gut-based bariatric surgery chronically elevates systemic bile acids and attenuates cocaine-induced elevations in accumbal dopamine. Notably, this surgery reduces reward-related behavior and psychomotor sensitization to cocaine. Utilizing a knockout mouse model, we have determined that a main mediator of these post-operative effects is the Takeda G protein-coupled bile acid receptor (TGR5). Viral restoration of TGR5 in the nucleus accumbens of TGR5 knockout animals is sufficient to restore cocaine reward, centrally localizing this TGR5-mediated modulation. These findings define TGR5 and bile acid signaling as pharmacological targets for the treatment of cocaine abuse and reveal a novel mechanism of gut-to-brain communication.

mTORC2/rictor signaling disrupts dopamine-dependent behaviors via defects in striatal dopamine neurotransmission.

Disrupted neuronal protein kinase B (Akt) signaling has been associated with dopamine (DA)-related neuropsychiatric disorders, including schizophrenia, a devastating mental illness. We hypothesize that proper DA neurotransmission is therefore dependent upon intact neuronal Akt function. Akt is activated by phosphorylation of two key residues: Thr308 and Ser473. Blunted Akt phosphorylation at Ser473 (pAkt-473) has been observed in lymphocytes and postmortem brains of schizophrenia patients, and psychosis-prone normal individuals. Mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multiprotein complex that is responsible for phosphorylation of Akt at Ser473 (pAkt-473). We demonstrate that mice with disrupted mTORC2 signaling in brain exhibit altered striatal DA-dependent behaviors, such as increased basal locomotion, stereotypic counts, and exaggerated response to the psychomotor effects of amphetamine (AMPH). Combining in vivo and ex vivo pharmacological, electrophysiological, and biochemical techniques, we demonstrate that the changes in striatal DA neurotransmission and associated behaviors are caused, at least in part, by elevated D2 DA receptor (D2R) expression and upregulated ERK1/2 activation. Haloperidol, a typical antipsychotic and D2R blocker, reduced AMPH hypersensitivity and elevated pERK1/2 to the levels of control animals. By viral gene delivery, we downregulated mTORC2 solely in the dorsal striatum of adult wild-type mice, demonstrating that striatal mTORC2 regulates AMPH-stimulated behaviors. Our findings implicate mTORC2 signaling as a novel pathway regulating striatal DA tone and D2R signaling.

Zn(2+) reverses functional deficits in a de novo dopamine transporter variant associated with autism spectrum disorder.

Our laboratory recently characterized a novel autism spectrum disorder (ASD)-associated de novo missense mutation in the human dopamine transporter (hDAT) gene SLC6A3 (hDAT T356M). This hDAT variant exhibits dysfunctional forward and reverse transport properties that may contribute to DA dysfunction in ASD. Here, we report that Zn(2+) reverses, at least in part, the functional deficits of ASD-associated hDAT variant T356M. These data suggest that the molecular mechanism targeted by Zn(2+) to restore partial function in hDAT T356M may be a novel therapeutic target to rescue functional deficits in hDAT variants associated with ASD.

Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors.

BACKGROUND: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. METHODS: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). OUTCOMES: Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis. INTERPRETATION: We determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.

PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein.

Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.

Missense dopamine transporter mutations associate with adult parkinsonism and ADHD.

Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.

Dopamine transporter deficiency syndrome: phenotypic spectrum from infancy to adulthood.

Dopamine transporter deficiency syndrome due to SLC6A3 mutations is the first inherited dopamine 'transportopathy' to be described, with a classical presentation of early infantile-onset progressive parkinsonism dystonia. In this study we have identified a new cohort of patients with dopamine transporter deficiency syndrome, including, most significantly, atypical presentation later in childhood with a milder disease course. We report the detailed clinical features, molecular genetic findings and in vitro functional investigations undertaken for adult and paediatric cases. Patients presenting with parkinsonism dystonia or a neurotransmitter profile characteristic of dopamine transporter deficiency syndrome were recruited for study. SLC6A3 mutational analysis was undertaken in all patients. The functional consequences of missense variants on the dopamine transporter were evaluated by determining the effect of mutant dopamine transporter on dopamine uptake, protein expression and amphetamine-mediated dopamine efflux using an in vitro cellular heterologous expression system. We identified eight new patients from five unrelated families with dopamine transporter deficiency syndrome. The median age at diagnosis was 13 years (range 1.5-34 years). Most significantly, the case series included three adolescent males with atypical dopamine transporter deficiency syndrome of juvenile onset (outside infancy) and progressive parkinsonism dystonia. The other five patients in the cohort presented with classical infantile-onset parkinsonism dystonia, with one surviving into adulthood (currently aged 34 years) and labelled as having 'juvenile parkinsonism'. All eight patients harboured homozygous or compound heterozygous mutations in SLC6A3, of which the majority are previously unreported variants. In vitro studies of mutant dopamine transporter demonstrated multifaceted loss of dopamine transporter function. Impaired dopamine uptake was universally present, and more severely impacted in dopamine transporter mutants causing infantile-onset rather than juvenile-onset disease. Dopamine transporter mutants also showed diminished dopamine binding affinity, reduced cell surface transporter, loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux. Our data series expands the clinical phenotypic continuum of dopamine transporter deficiency syndrome and indicates that there is a phenotypic spectrum from infancy (early onset, rapidly progressive disease) to childhood/adolescence and adulthood (later onset, slower disease progression). Genotype-phenotype analysis in this cohort suggests that higher residual dopamine transporter activity is likely to contribute to postponing disease presentation in these later-onset adult cases. Dopamine transporter deficiency syndrome remains under-recognized and our data highlights that dopamine transporter deficiency syndrome should be considered as a differential diagnosis for both infantile- and juvenile-onset movement disorders, including cerebral palsy and juvenile parkinsonism.

Structural arrangement of the intracellular Ca2+ binding domains of the cardiac Na+/Ca2+ exchanger (NCX1.1): effects of Ca2+ binding.

The cardiac Na(+)/Ca(2+) exchanger (NCX1.1) serves as the primary means of Ca(2+) extrusion across the plasma membrane of cardiomyocytes after the rise in intracellular Ca(2+) during contraction. The exchanger is regulated by binding of Ca(2+) to its intracellular domain, which contains two structurally homologous Ca(2+) binding domains denoted as CBD1 and CBD2. NMR and x-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have shown how Ca(2+) binding affects their structures and motional dynamics. However, structural information on the entire Ca(2+) binding domain, denoted CBD12, and how binding of Ca(2+) alters its structure and dynamics is more limited. Site-directed spin labeling has been employed in this work to address these questions. Electron paramagnetic resonance measurements on singly labeled constructs of CBD12 have identified the regions that undergo changes in dynamics as a result of Ca(2+) binding. Double electron-electron resonance (DEER) measurements on doubly labeled constructs of CBD12 have shown that the ß-sandwich regions of the CBD1 and CBD2 domains are largely insensitive to Ca(2+) binding and that these two domains are widely separated at their N and C termini. Interdomain distances measured by DEER have been employed to construct structural models for CBD12 in the presence and absence of Ca(2+). These models show that there is not a major change in the relative orientation of the two Ca(2+) binding domains as a result of Ca(2+) binding in the NCX1.1 isoform. Additional measurements have shown that there are significant changes in the dynamics of the F-G loop region of CBD2 that merit further characterization with regard to their possible involvement in regulation of NCX1.1 activity.

Exendin-4 decreases amphetamine-induced locomotor activity.

Glucagon-like peptide-1 (GLP-1) is released in response to nutrient ingestion and is a regulator of energy metabolism and consummatory behaviors through both peripheral and central mechanisms. The GLP-1 receptor (GLP-1R) is widely distributed in the central nervous system, however little is known about how GLP-1Rs regulate ambulatory behavior. The abused psychostimulant amphetamine (AMPH) promotes behavioral locomotor activity primarily by inducing the release of the neurotransmitter dopamine. Here, we identify the GLP-1R agonist exendin-4 (Ex-4) as a modulator of behavioral activation by AMPH. We report that in rats a single acute administration of Ex-4 decreases both basal locomotor activity as well as AMPH-induced locomotor activity. Ex-4 did not induce behavioral responses reflecting anxiety or aversion. Our findings implicate GLP-1R signaling as a novel modulator of psychostimulant-induced behavior and therefore a potential therapeutic target for psychostimulant abuse.

Dysregulation of dopamine transporters via dopamine D2 autoreceptors triggers anomalous dopamine efflux associated with attention-deficit hyperactivity disorder.

The neurotransmitter dopamine (DA) modulates brain circuits involved in attention, reward, and motor activity. Synaptic DA homeostasis is primarily controlled via two presynaptic regulatory mechanisms, DA D(2) receptor (D(2)R)-mediated inhibition of DA synthesis and release, and DA transporter (DAT)-mediated DA clearance. D(2)Rs can physically associate with DAT and regulate DAT function, linking DA release and reuptake to a common mechanism. We have established that the attention-deficit hyperactivity disorder-associated human DAT coding variant Ala559Val (hDAT A559V) results in anomalous DA efflux (ADE) similar to that caused by amphetamine-like psychostimulants. Here, we show that tonic activation of D(2)R provides support for hDAT A559V-mediated ADE. We determine in hDAT A559V a pertussis toxin-sensitive, CaMKII-dependent phosphorylation mechanism that supports D(2)R-driven DA efflux. These studies identify a signaling network downstream of D(2)R activation, normally constraining DA action at synapses, that may be altered by DAT mutation to impact risk for DA-related disorders.

Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

Currents in response to rapid concentration jumps of amphetamine uncover novel aspects of human dopamine transporter function.

Amphetamine (AMPH) is a widely abused psychostimulant that acts as a substrate for the human dopamine transporter (hDAT). Using a piezoelectric rapid application system, we measured AMPH-induced currents mediated by hDAT. Whole-cell patch-clamp recordings in a heterologous expression system reveal that AMPH induces a rapidly activating and subsequently decaying inward current mediated by hDAT. We hypothesize that this transient inward current reflects a conformational change associated with substrate translocation. The AMPH-induced current strictly depends on extracellular Na+. Elevated intracellular Na+ has no effect on the peak AMPH-induced current amplitude but inhibits the steady-state current. In addition, elevated intracellular Na+ causes an overshoot outward current upon washout of AMPH that reflects hDAT locked in a Na+-exchange mode. Furthermore, elevated intracellular Na+ dramatically accelerates the recovery time from desensitization of the AMPH-induced current, revealing a new role for intracellular Na+ in promoting the transition to the hDAT "outward-facing" conformation. Ion substitution suggests that both extracellular and intracellular Cl- facilitate transporter turnover in contrast to the classical model of Cl- as a cotransported ion. We present an alternating-access model of hDAT function that accurately fits the main features of the experimental data. The model predicts that translocation of substrate occurs within milliseconds of substrate binding but that slow reorientation of the empty transporter is the rate-limiting factor for turnover. The model provides a framework for interpreting perturbations of hDAT activity.

Zinc inhibition of rat NR1/NR2A N-methyl-D-aspartate receptors.

Zinc ions (Zn(2+)) are localized in presynaptic vesicles at glutamatergic synapses and released in an activity-dependent manner. Modulation of NMDA-type glutamate receptors by extracellular Zn(2+) may play an important role under physiological conditions and during pathologies such as ischaemia or seizure. Zn(2+) inhibits NMDA receptors containing the NR2A subunit with an IC(50) value in the low nanomolar concentration range. Here we investigate at the single-channel level the mechanism of high affinity Zn(2+) inhibition of recombinant NR1/NR2A receptors expressed in HEK293 cells. Zn(2+) reversibly decreases the mean single-channel open duration and channel open probability determined in excised outside-out patches, but has no effect on single-channel current amplitude. A parallel series of experiments demonstrates that lowering extracellular pH (increasing proton concentration) has a similar effect on NR1/NR2A single-channel properties as Zn(2+). Fitting the sequence of single-channel events with kinetic models suggests that the association of Zn(2+) with its binding site enhances proton binding. Modelling further suggests that protonated channels are capable of opening but with a lower open probability than unprotonated channels. These data and analyses are consistent with Zn(2+)-mediated inhibition of NMDA receptors primarily reflecting enhancement of proton inhibition.