Functional aspects of cloned neurotransmitter transporters will be studied in heterologous expression systems, with emphasis on the rat serotonin transporter rSERT and the rat GABA transporter rGAT1. It is scientifically wise and efficient to intermix studies on these two homologous transporters (~ 40% primary sequence similarity). The first specific aims probe these themes of structure and function with established techniques: single-channel measurements, including simulations; proton permeation; functional characteristics of coexpressed mutants; substituted cysteine mutagenesis; and localization of the gates. The second set of specific aims probes these same themes, with innovative techniques that are obviously riskier but that may provide novel information: biocytin labeling with the nonsense suppression technique; photolytic cleavage, also with unnatural amino-acid residues; fluorescence assays of conformational changes during function. Novel proteins will be sought that interact with the transporters, using the yeast two-hybrid system. Predictions about the timing of substrate flux after a pulse of neurotransmitter will be tested using time-resolved amperometry. Fluoxetine typifies a class of antidepressants, introduced in the past decade, called serotonin-selective reuptake inhibitors (SSRI's). Many patients diagnosed with attention-deficit disorder (ADD) or attention deficit-hyperactivity disorder (ADHD) benefit from methylphenidate, amphetamines, or other agents thought to act on monoamine transporters either by blocking uptake or by enhancing release. Therefore neurotransmitter transporters deserve continued close study with modern molecular techniques.
Showing the most recent 10 out of 20 publications
