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Uptake blocker-induced conformational changes of the dopamine transporter revealed by proteolysis.

Introduction. The dopamine transporter (DAT) is a membrane bound protein responsible for the clearance of dopamine from the synapse. Psychostimulant drugs such as cocaine and methamphetamine target DAT resulting in increased synaptic levels of dopamine. Understanding the mechanism of action for uptake blockers such as cocaine will aid in the design of novel therapies for drug addiction and neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. DAT is a membrane-bound, polytopic, glycoprotein protein that co-transports dopamine, [Na.sup.+], and [Cl.sup.-]. Computer generated models predict twelve transmembrane domains (TMs) with intracellular N- and C-termini. Current experimental evidence suggests the TM domains are critical for uptake blocker binding and substrate transport, however relatively little is known about the role of the extracellular loops (ELs) in these processes. EL2 is a large 78 amino acid loop connecting TM3 and TM4, a region associated with uptake blocker binding and substrate translocation. In this study we examine the role of EL2 in uptake blocker and substrate binding by monitoring its sensitivity to proteolysis.

Methods. Membranes or synaptosomes were prepared from rat striatum, treated with trypsin or Asp-N in the presence or absence of blockers/substrates, and DAT proteolysis analyzed by SDS-PAGE and immunoblotting. To examine the effect of EL2 proteolysis on binding membranes were digested with protease and incubated on ice with [[sup.3]H]CFT, a cocaine analog, for 1 hour. Similarly, to examine the effect of proteolysis on uptake synaptosomes were digested with protease, and incubated with [[sup.3]H]dopamine for 5 minutes at 37[degrees]C in modified Krebs-phosphate buffer containing.

Results. Proteolysis of DAT with trypsin produces a 45 kDa glycosylated fragment visualized by immunoblotting with mAb 16. This fragment is consistent with proteolysis at R218 located on the C-terminal region of EL2. In the presence of DAT uptake blockers but not substrates DAT sensitivity to trypsin is reduced 100-1000 fold. Blocker-induced protease resistance was [Na.sup.+]-dependent and was not observed in the presence of imipramine or desipramine, serotonin (SERT) and norepinephrine transporter (NET) blockers, indicating it was specific for uptake blocker binding. Striatal membranes or synaptosomes proteolyzed with trypsin display decreases in [[sup.3]H]CFT binding and [[sup.3]H]dopamine transport, suggesting the integrity of EL2 is required for binding and transport activity. Proteolysis with Asp-N produces 20 and 21 kDa fragments when immunoblotted with mAb16. These fragments correspond to potential protease sites at D174, D191, or D199, located in the N-terminal region of EL2. As observed with trypsin, DAT uptake blockers but not substrates reduced Asp-N proteolysis by at least 30%. Digestion of EL2 with Asp-N prior to [[sup.3]H]CFT binding and [[sup.3]H]dopamine uptake resulted in a loss of DAT function consistent with the extent of proteolysis as monitored by immunoblotting.

Discussion. These data provide evidence for conformational changes that occur in DAT during binding of uptake blockers but not substrates. Uptake blocker-induced protease resistance is apparent in both N- and C-terminal regions of EL2 implying that substantial conformational changes occur over a large portion of EL2. All uptake blockers tested affected the protease sensitivity of EL2 similarly, supporting the hypothesis that they use a single overlapping binding pocket. Moreover, this data also implies uptake blockers are not static molecules that simply block a permeation pathway, but instead actively induce movements in DAT. The finding that substrates do not induce protease resistance in EL2 indicates they may utilize a separate binding mechanism distinguishable from blockers. This study provides novel data for the role of EL2 in DAT function.

This work is supported by F31 DA 14857-01

Jon D. Gaffaney * and Roxanne A. Vaughan

Department of Biochemistry and Molecular Biology. University of North Dakota School of Medicine and Health Sciences, Grand Forks, N.D.
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Title Annotation:A. Rodger Denison Student Research Competition: COMMUNICATIONS: GRADUATE DIVISION
Author:Gaffaney, Jon D.; Vaughan, Roxanne A.
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2004
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