Uptake Inhibitors but not Substrates Induce Protease Resistance in Extracellular Loop Two of the Dopamine Transporter

Abstract

Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 g/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2␤-carbomethoxy-3␤-(4-flourophenyl)tropane (␤-CFT), nomifensine, benztropine, or (Ϫ)-cocaine, 100-to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn 2ϩ , (ϩ)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [ H]CFT and [3 H]dopamine. Digestion decreased the B max for binding and the V max for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.

Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics

“…Materials- [7,8-3 H]DA (45 Ci/mmol) was from PerkinElmer Life Sciences; [9, H]palmitic acid (73.4 Ci/mmol) was from Moravek; H 3 32 PO 4 was from MP Biomedicals; Fluoro-Hance fluorographic reagent was from Research Products International; DA was from Research Biochemicals International; phorbol 12-myristate,13-acetate (PMA), oleoyl-2-acetyl-snglycerol (OAG), and okadaic acid (OA) were from EMD Millipore; DAT polyclonal antibody 16 (poly16) and monoclonal antibody 16 (mAb 16) were as previously described (23,24); polyclonal antibodies for Rab5A, Rab7, and Na ϩ /K ϩ ATPase were from Santa Cruz Biotechnology; those for Rab11 were from BD Biosciences, and for HA were from Covance; X-tremeGENE HP transfection reagent was from Roche Applied Bioscience; methyl methanethiosulfonate (MMTS), HPDP biotin (sulfhydryl-reactive (N-(6-(biotinamido)hexyl)-3Ј-(2Ј-pyridyldithio)-propionamide), sulfo-NHS-SS-biotin, high capacity NeutrAvidin-agarose resin, protease inhibitor tablets, and bicinchoninic acid protein assay reagent were from Thermo Scientific; PitStop2® was from Abcam Biochemicals; (Ϫ)-cocaine and other fine chemicals were from Sigma. Rats were purchased from Charles River Laboratories.…”

“…Lysates were centrifuged at 4,000 ϫ g for 2 min, and resulting supernatants were adjusted to contain 0.5% SDS and centrifuged at 20,000 ϫ g for 30 min to remove insoluble material. DATs were immunoprecipitated with poly16 followed by SDS-PAGE and autoradiography (19,23). For experiments in brain tissue, rat striatal synaptosomes were prepared and labeled with 32 P as previously described (25).…”

Labeling of Dopamine Transporter Transmembrane Domain 1 with the Tropane Ligand N-[4-(4-Azido-3-[125I]iodophenyl)butyl]-2β-carbomethoxy-3β-(4-chlorophenyl)tropane Implicates Proximity of Cocaine and Substrate Active Sites

“…Pooled stable cell lines were maintained at 37°C in complete medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin) supplemented with 250 g/ml G418 in an incubator gassed with 5% CO 2 /95% O 2 . Expression of mutants was verified by immunoblotting with hDAT-specific antibody (MAb 369; Millipore Bioscience Research Reagents, Temecula, CA) (Gaffaney and Vaughan, 2004). All mutants were assayed for cocaine-displaceable [ 3 H]DA uptake and binding of the cocaine analog 2␤-carbomethoxy-3␤-(4-fluorophenyl)tropane ([ 3 H]CFT) as described previously (Gaffaney and Vaughan, 2004;Vaughan et al, 2007).…”

“…Expression of mutants was verified by immunoblotting with hDAT-specific antibody (MAb 369; Millipore Bioscience Research Reagents, Temecula, CA) (Gaffaney and Vaughan, 2004). All mutants were assayed for cocaine-displaceable [ 3 H]DA uptake and binding of the cocaine analog 2␤-carbomethoxy-3␤-(4-fluorophenyl)tropane ([ 3 H]CFT) as described previously (Gaffaney and Vaughan, 2004;Vaughan et al, 2007).…”

Distinctions between Dopamine Transporter Antagonists Could be Just around the Bend: Fig. 1.

“…Loland et al (2008) tackle this issue with molecular approaches that can define conformational states in DAT before and after antagonist binding. The adoption of distinct conformational changes in DAT upon antagonist binding compared with DA-bound DAT has already been suggested using protease sensitivity assays (Gaffaney and Vaughan, 2004). Now, Loland et al (2008) probe conformational changes in DAT, both before and after binding of the BZTs, by monitoring accessibility of an introduced cysteine at residue Ile159 to cysteine-reactive methanethiosulfonates (MTS).…”