Uptake blocker-induced conformational changes of the dopamine transporter revealed by 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.
|Printer friendly Cite/link Email Feedback|
|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|
|Date:||Apr 1, 2004|
|Previous Article:||Psychostimulant and substrate-induced dopamine transporter phosphorylation: a transport and protein kinase C dependent mechanism.|
|Next Article:||Sexually transmitted HPV serotype variations resulting in high-grade cervical dysplasia in North-East North Dakota and North-West Minnesota.|