Abstract
Determining the contributions of different transporter species to overall cellular transport is fundamental for understanding the physiological regulation of solutes. We calculated the relative activities of Solute Carrier (SLC) transporters using the Michaelis-Menten equation and global fitting to estimate the normalized maximum transport rate for each transporter (Vmax). Data input were the normalized measured uptake of the essential neutral amino acid (AA) L-leucine (Leu) from concentration-dependence assays performed using Xenopus laevis oocytes. Our methodology was verified by calculating Leu and L-phenylalanine (Phe) data in the presence of competitive substrates and/or inhibitors. Among 9 potentially expressed endogenous X. laevis oocyte Leu transporter species, activities of only the uniporters SLC43A2/LAT4 (and/or SLC43A1/LAT3) and the sodium symporter SLC6A19/B0AT1 were required to account for total uptake. Furthermore, Leu and Phe uptake by heterologously expressed human SLC6A14/ATB0,+ and SLC43A2/LAT4 was accurately calculated. This versatile systems biology approach is useful for analyses where the kinetics of each active protein species can be represented by the Hill equation. Furthermore, its applicable even in the absence of protein expression data. It could potentially be applied, for example, to quantify drug transporter activities in target cells to improve specificity.
Highlights
In this study we used the Xenbase microarray data to determine the set of potentially expressed xAAT species in unfertilized oocytes
This search identified 9 Solute Carrier (SLC) genes that code for Leu xAATs with mRNA expression (Table 1)
Since there are no reported kinetic studies for xAAT species, Km values used in the calculations were taken from published reports of mammalian orthologs assayed by heterologous expression in X. laevis oocytes (Table 1)
Summary
Our aims were to develop a strategy to (1) quantify the relative function of specific SLC species within a system of transporters with a variety of substrate affinities and transport mechanisms, and (2) experimentally verify the calculations for total transport and for responses to new stimuli by individual transporter species
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