There is mounting evidence that the choline cotransporter (ChCoT) cycles into and out of the plasma membrane via a series of endosomal compartments. Endosomal ablation revealed a difference in the intracellular compartments required for constitutively cycling versus regulated glucose facilitative transporter cycling. Tissue exposed to transferrin conjugated to horseradish peroxidase followed by treatment with 3, 3'-diaminobenzidine with hydrogen peroxide will be subjected to presumed ablation. In ablated Limulus brain hemi-slices [3H]choline transport was reduced by more than 70%. However, antecedent elevated potassium treatment of ablated slices restored transport. Our working hypothesis is that the ChCoT cycles continuously into and out of the plasma membrane via established early endosomal compartments. A secondary hypothesis is that direct phosphorylation of the ChCoT in the plasma membrane targets it for lysosomal degradation subsequent to endocytotic removal from the membrane. Further, we hypothesize that the activity driven regulated ChCoT trafficking involves an endosomal compartment separate from the early endosomal compartments essential for constitutive transporter trafficking. An understanding of the factors determining the trafficking paradigms of the ChCoT is necessary to elucidate the physiological regulation of this transporter.
We aim to test the above hypotheses by the following set of Specific Aims: 1. To determine, by cell surface biotinylation strategies, the intracellular trafficking pathway subserving the constitutive cycling of the cholinergic choline cotransporter. 2. To determine the role of direct phosphorylation of the cotransporter on the trafficking pathway. 3. To determine, by compartmental ablation strategies, the intracellular pathway unique to activitydependent regulated cotransporter trafficking.
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