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 Ϫ60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 M) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at Ϫ60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca 2ϩ /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.

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Rapid treatment (1 min) of rat striatal synaptosomes with low-dose amphetamine increases surface expression of the dopamine transporter (DAT). Using mouse neuroblastoma N2A cells, stably transfected with green fluorescent protein-DAT, we demonstrate the realtime substrate-induced rapid trafficking of DAT to the plasma membrane using total internal reflection fluorescence microscopy (TIRFM). Both the physiological substrate, dopamine, and amphetamine began to increase surface DAT within 10 s of drug addition and steadily increased surface DAT until removal 2 min later. The substrate-induced rise in surface DAT was dose-dependent, was blocked by cocaine, and abated after drug removal. Although individual vesicle fusion was not visually detectable, exocytosis of DAT was blocked using both tetanus neurotoxin and botulinum neurotoxin C to cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Notably, the dopamine-induced increase in surface DAT was cocaine-sensitive but D 2 -receptor independent. TIRFM data were confirmed in human DAT-N2A cells using biotinylation, and similar effects were detected in rat striatal synaptosomes. A specific inhibitor of protein kinase C-␤ blocked the substrate-mediated increase in surface DAT in both DAT-N2A cells and rat striatal synaptosomes. These data demonstrate that the physiological substrate, dopamine, and amphetamine rapidly increase the trafficking of DAT to the surface by a mechanism dependent on SNARE proteins and protein kinase C-␤ but independent of dopamine D 2 receptor activation. Importantly, this study suggests that the reuptake system is poised to rapidly increase its function during dopamine secretion to tightly regulate dopaminergic neurotransmission.

The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane Nterminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [ 3 H]dopamine uptake but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn 2ϩ markedly potentiated [ 3 H]dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward-and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [ 3 H]dopamine uptake and [ 3 H]dopamine efflux was consistent with a slowed transition between inward-and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a threedimensional molecular model of DAT.The strength and duration of dopaminergic neurotransmission is tightly controlled by the dopamine transporter (DAT), which mediates reuptake of synaptic dopamine (DA) (Chen and Reith, 2000) and is a target for therapeutic drugs and abused psychostimulants (Sulzer et al., 2005). Amphetamine (AMPH), a substrate for DAT, competitively inhibits DA reuptake and elicits outward transport of DA by reversal of the transporter (Sulzer et al., 2005), purportedly by an exchange diffusion mechanism (Fischer and Cho, 1979;Sulzer et al., 2005). This model assumes that the transporter transitions between two primary conformational states-an "outwardfacing" conformation favoring substrate binding and influx, and an "inward-facing" conformation in which the binding sites are available to the intracellular milieu and which favors substrate efflux. In the absence of substrate, the transporter favors an outward-facing conformation ready to bind

The dopamine transporter (DAT) is a key mediator of dopaminergic neurotransmission and a major target for amphetamine. We found previously that protein kinase C (PKC) ␤ regulates amphetamine-mediated dopamine efflux. Here, using PKC␤ wild-type (WT) and knockout (KO) mice, we report a novel role for PKC␤ in amphetamine-induced regulation of DAT trafficking and activity. PKC␤ KO mice have less striatal surface DAT, [ 3 H]dopamine uptake, and amphetamine-stimulated dopamine efflux, yet higher novelty-induced locomotor activity than WT mice. Although a short exposure (Յ90 s) to amphetamine rapidly increases striatal surface DAT and [3 H]dopamine uptake in WT mice, this treatment decreases surface DAT and [3 H]dopamine uptake in KO mice. Increases in surface DAT and [ 3 H]dopamine uptake are not evident in KO mice until a longer exposure (60 min) to amphetamine, by which time WT mice exhibit decreased surface DAT and dopamine uptake. The slowness of amphetamine-induced striatal DAT trafficking in PKC␤ KO mice was mimicked by the use of a specific PKC␤ inhibitor, LY379196, in WT mice. Furthermore, PKC␤ KO mice exhibit reduced locomotor responsiveness to amphetamine compared with WT, which could be explained by reduced surface DAT and delayed amphetamine-induced DAT trafficking in KO mice. Our results indicate that PKC␤ is crucial for proper trafficking of DAT to the surface and for functioning of DAT and amphetamine signaling, providing new insight into the role of PKC␤ as an important regulator of dopaminergic homeostasis.The dopamine transporter (DAT) is an integral membrane protein containing 12 putative transmembrane domains and cytosolic amino and carboxyl termini. It belongs to a superfamily of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters. Dopaminergic transmission in the synapse is primarily terminated by removal of dopamine (DA) from presynaptic nerve terminals via DAT. DAT is also a primary target of psychostimulants such as amphetamine (AMPH). As a substrate, AMPH elicits reverse transport of DA through DAT, a mechanism underlying AMPH addiction. Dysfunctional DAT signaling is also implicated in various other neurodegenerative and psychiatric disorders such as Parkinson's disease, attention deficit/hyperactive disorder, and schizophrenia.DAT trafficking and activity are regulated by a complicated protein network involving multiple protein kinases and DAT-interacting proteins (Torres, 2006), among which protein kinase C (PKC) is the most characterized. Longer term activation of PKC (Ͼ20 min) by phorbol 12-myristate 13-acetate, a general PKC activator, leads to increased DAT endocytosis and reduced DAT recycling, resulting in reduced surface DAT expression and activity in rat synaptosomes and cultured cells (Daniels and Amara, 1999;Melikian and Buckley, 1999;Loder and Melikian, 2003). In accordance, PKC inhibitors block phorbol 12-myristate 13-acetate-induced DAT internalization (Daniels and Amara, 1999;Melikian and Buckley, 1999;Chang et al., 2001). An endocytic motif in the C terminus of DAT t...

Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition and gastroenteritis as well as posing a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and > 500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An x-ray crystal structure of a representative E•IMP•inhibitor complex is also presented. Overall, the secondary amine derivative 15a (Q67) demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines.

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10(3) selectivity for the parasite enzyme over human IMPDH2.