The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-⌬N-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-⌬N-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60 -65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the "outward facing" DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-⌬N-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.

The plasma membrane dopamine (DA) transporter (DAT) is essential for reuptake of extracellular DA. DAT function in heterologous cells is regulated by subcellular targeting, endocytosis, and intracellular trafficking, but the mechanisms regulating neuronal DAT remain poorly understood. Hence, we generated a knock-in mouse expressing a hemagglutinin (HA)-epitope-tagged DAT to study endogenous transporter trafficking. Introduction of the HA tag into the second extracellular loop of mouse DAT did not perturb its expression level, distribution pattern, or substrate uptake kinetics. Live-cell fluorescence microscopy imaging using fluorescently labeled HA-specific antibody and a quantitative HA-antibody endocytosis assay demonstrated that in axons HA-DAT was primarily located in the plasma membrane and internalized mostly in growth cones and varicosities, where synaptic vesicle markers were also concentrated. Formation of varicosities was frequently preceded or accompanied by highly dynamic filopodia-like membrane protrusions. Remarkably, HA-DAT often concentrated at the tips of these filopodia. This pool of HA-DATs exhibited low lateral membrane mobility. Thus, DAT-containing filopodia may be involved in synaptogenesis in developing DA neurons. Treatment of neurons with amphetamine increased mobility of filopodial HA-DAT and accelerated HA-DAT endocytosis in axons, suggesting that chronic amphetamine may interfere with DA synapse development. Interestingly, phorbol esters did not accelerate endocytosis of axonal DAT.

alpha(S1)-Casein is one of the major protein components of the casein fraction of milk. Curcumin (diferuloyl methane), the major curcuminoid, constituting about 2-5% of turmeric (Curcuma longa ) is the active ingredient with many physiological, biochemical, and pharmacological properties. On the basis of spectroscopic measurements, it is inferred that curcumin binds to alpha(S1)-casein at pH 7.4 and 27 degrees C with two binding sites, one with high affinity [(2.01 +/- 0.6) x 10(6) M(-1)] and the other with low affinity [(6.3 +/- 0.4) x 10(4) M(-1)]. Binding of curcumin to alpha(S1)-casein is predominantly hydrophobic in nature. The anisotropy of curcumin or conformation of alpha(S1)-casein did not change on interaction. The stability of curcumin in solution at pH 7.2 was enhanced on binding with alpha(S1)-casein. The chaperone-like activity of alpha(S1)-casein gets slightly enhanced on its binding to curcumin. The ability of curcumin to protect erythrocytes against hemolysis was not affected due to curcumin- alpha(S1)-casein interaction. The two binding sites of alpha(S1)-casein for curcumin, along with enhanced solution stability on interaction, may offer an alternative approach in physiological and nutritional applications.

Dopamine (DA) transporter (DAT) functions at the surface of dopaminergic neurons to clear extracellular DA. DAT surface levels are regulated by endocytosis. However, the endosomelysosome system is not well characterized in dopaminergic neurons and the endocytic trafficking of endogenous DAT is poorly studied. Hence we analyzed the distribution of endocytic compartments and DAT localization in cultured rat embryonic and postnatal neurons using fluorescence microscopy. Early Rab5 and EEA.1 containing endosomes were mostly found in somatodendritic regions of neurons, whereas endosomes containing recycling markers were primarily found in axons. In axons, DAT was located mainly in recycling endosomes and plasma membrane whereas in cell bodies and dendrites DAT was detected in early, late and recycling endosomal compartments. Subcellular fractionation of adult rat striatal synaptosomes demonstrated that DAT is enriched in fractions containing plasma membrane and recycling endosomes. This pattern of DAT distribution was not altered upon activation of protein kinase C in postnatal DA neurons. Altogether, our data suggest that axonal DAT mainly shuttles between the plasma membrane and recycling endosomes, whereas in the somatodendritic region of neurons DAT traffics through all conventional endosomal pathways.