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Utah's Foremost Platform for Undergraduate Research Presentation
2022 Abstracts

Exploration of Michaelis-Menten kinetics of the dopamine transporter in the striatum using iontophoresis

Presenters: Ali Allred ; Matthew Burris
Authors: Ali Allred, Matthew Burris, Joakim Ronström, Emma Read, Brayden Tolman, Anna Everett, Hillary Wadsworth, Jared Willets, Eliza White, Matthew Rasmussen, Jordan Yorgason
Faculty Advisor: Jordan Yorgason
Institution: Brigham Young University

The main objective of this study is to investigate the Michaelis-Menten (MM) kinetics for the dopamine transporter (DAT) in the striatum and the effect of psychostimulants on DAT trafficking. Traditionally, the Michaelis-Menten (MM) enzymatic model is used to determine the maximal rate of uptake (Vmax) of dopamine (DA) by the DAT as well as provide information on the enzymatic binding activity (Km). The MM parameters for the DAT have been examined and characterized previously, albeit under non-physiological conditions that may not be reflective of actual DAT activity. First, previous experiments determining MM Kmparameters have used homogenized tissue. The preparation of homogenized tissue is crude and disrupts ion gradients, intracellular organelles, such as mitochondria, and overall cellular function. Thus, previous estimates of MM parameters based on these conditions may be adversely affected by these non-physiological conditions. Second, voltammetry studies measuring evoked dopamine release have assumed DAT saturation for determining Vmaxwithout proving saturation under electrically stimulated release conditions. The assumption for DAT saturation is again based partly on Kmvalues from homogenized tissue studies, which estimate Kmvalues at ~160-200 nM. Furthermore, DAT Vmaxvalues from MM modeling studies correlate with release amplitude, suggesting that DATs are undersaturated and MM conditions are being violated for some of these previous studies. The validity of these parameters was investigated using exogenously application of DA (iontophoresis and puff application) in the striatum in adolescent mice. The DAT was saturated by applying exogenous DA with increasing concentrations, while measuring uptake. The Vmaxand Kmparameters were then calculated from these experiments. To quantify the effect of psychostimulants on these MM parameters, the same procedure was conducted using increasing concentrations of cocaine and GBR12909—two drugs known to inhibit DAT activity.