XB-ART-52954Sci Rep. March 28, 2017; 7 40628.
Quantifying the relative contributions of different solute carriers to aggregate substrate transport.
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/B(0)AT1 were required to account for total uptake. Furthermore, Leu and Phe uptake by heterologously expressed human SLC6A14/ATB(0,+) 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.
PubMed ID: 28091567
PMC ID: PMC5238446
Article link: Sci Rep.
Article Images: [+] show captions
|Figure 1. Diagram of the method used for quantifying activity of specific transporter species.The left panel is a schematic of the steps taken in the current study to establish and verify the method for amino acid transport in Xenopus laevis oocytes. AAT refers to Solute Carrier amino acid transporters for L-leucine and L-phenylalanine. The source for the mRNA expression data was Xenbase as described in Methods and Results. Uptake assay protocols are described in Methods. Defined uptake buffers are described in Methods, Results, and Fig. 2. The input parameters for the calculations are given in Table 1. The rationale for the specific equations used to construct the current model are given in the methods. The right panel is a generalized series of steps that could be applied to any system where the kinetics of each active enzyme species can be represented by the Hill equation.|
|Figure 2. Inhibition of L-leucine transporters in tested uptake buffers.L-leucine (Leu) uptake was tested in buffers containing 10 mM added competitors (BCH or amino acids). The Leu transporters (AATs) with affinity (Km (mM) values shown in Table 1) for the added competitors are indicated in the filled cells of the same color as the text for the named competitors. For Na+free uptake buffers, the Na+-dependent Leu AATs (i.e. require Na+ for Leu uptake) are indicated by ND. Additionally, responses of the Leu transporters for the competitor listed first for each uptake buffer are shown in the upper row and for the competitor listed second are shown in the lower row of filled cell(s). Clear boxes indicate AAT Leu uptake is unchanged due to addition of the indicated competitor and are used for AATs that transport Leu with affinities listed in Table 1 but do not transport the added competitor. aKm (mM) value is for low affinity kinetic component.|
|Figure 3. L-leucine uptake by endogenous Xenopus laevis oocyte transporters.Three minute uptakes by non-injected (NI) oocytes of 10–1000 μM L-leucine (Leu) in uptake buffers containing (+Na+) and without sodium (Na+free) were tested using radiolabeled amino acid (AA) tracers. In all panels uptake data (pmol/3 min per oocyte) were normalized to uptake in 1 mM Leu, +Na+ uptake buffer for each batch of oocytes and experimental day. Panel (A) shows normalized Leu uptake rate data (expt) vs. the simultaneous fit (fit) for endogenous Leu uptake rates. Panel (B) shows model calculations (model) for contributions to uptake of 10–1000 μM Leu by various endogenous Xenopus laevis oocyte (xAAT) species. Model output was calculated based on the calculated xAAT Vmax,i and the reported Km for each xAAT ortholog (Table 1). The 95% confidence limits for transporter activities are shown (dotted lines) bracketing the model predictions. Panels (C,D) show the calculated Leu uptake rates in +Na+ and Na+ free uptake buffers containing excess competitive inhibitors. Panel (C) shows the concentration dependence (0–1000 μM Leu) of normalized cumulative uptake data by NI oocytes in Na+free uptake buffers containing 10 mM each of the following competitors: L-alanine (Ala) (Na+free+Ala), Ala and L-tryptophan (Trp) (Na+free+Ala+Trp), L-valine (Val) (Na+free+Val) vs. calculated cumulative endogenous uptake results for the respective uptake buffers. For uptakes in Na+free+Ala, and Na+free+Ala+Trp uptake buffers, the calculated values were virtually indistinguishable from the global fit for the Na+free data, therefore a single line was used for graphing all three data sets. Panel (D) shows the concentration dependence (0–1000 μM) of total Leu uptake rates in +Na+ uptake buffers containing 10 mM each of the following competitors: Ala, Arg (+Na++Ala+Arg), 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) (+Na++BCH), and BCH and Ala (+Na++BCH+Ala) (Fig. 2). n = 6–8 ooyctes each experiment, for 3 independent experiments. The experimental data are shown as the mean ± SEM for the measured uptake rates and the calculated cumulative endogenous uptake rates were based on the previously calculated Vmax and reported Km values for the kinetic components exhibited by each AAT species (Table 1).|
|Figure 4. L-leucine and L-phenylalanine uptake by endogenous Xenopus laevis oocyte and exogenously expressed human sodium-dependent symporter SLC6A14/ATB0,+ and sodium-independent uniporter SLC43A2/LAT4 transporters.L-Leucine (Leu) uptake rates (pmol/3 min per oocyte) from non-injected (NI) and oocytes expressing human ATB0,+ (hATB0,+) were compared with model calculations. Measured uptake data (pmol/10 min per oocyte) for each batch of oocytes and experimental day was normalized to uptake rates by NI oocytes in 1 mM Leu, +Na+ uptake buffer. Panel (A) shows measured Leu (10–3000 μM) uptake rates for NI oocytes (NI, expt), and for oocytes expressing hATB0,+ without subtraction of NI uptake data (NI+hATB0,+, expt) vs. the calculated model outputs for the respective oocytes (NI, model; and NI+ATB0+, model). Panel (B) shows experimental data for total Leu (0–3000 uM) uptake rates by hATB0,+ expressing oocytes with subtraction of NI oocyte uptake rates (+hATB0,+, expt) vs. calculated Leu uptake by endogenous Xenopus laevis oocyte Leu transporters (xB0AT1, model; and xLAT4, model) and exogenous hATB0,+ transporters (+hATB0,+, model). n = 6–8 oocytes per experiment for 7 independent experiments. Panels (C,D) show L-phenylalanine (Phe) uptake data (pmol/10 min per oocyte) in Na+ containing uptake buffer vs. model calculations for endogenous Phe transporters (xB0AT1, xLAT4, xTAT1) and human LAT4 (hLAT4) transporters. Experimental uptake (pmol/10 min per oocyte) for all oocytes was normalized to uptake in 10 mM Phe, Na+ containing- uptake buffer (Na+). Panel (C) shows normalized total Phe uptake rates by NI oocytes (NI, expt) vs. oocytes with expressed hLAT4 without subtraction of NI oocyte uptake (NI+hLAT4, expt) vs. calculated data for the respective oocyte uptakes (NI, model; and NI+hLAT4, model). Panel (D) shows the normalized data for 0–10 mM Phe uptake rates by oocytes exogenously expressing hLAT4 with subtraction of NI uptake (hLAT4, expt) vs. model calculations for hLAT4 (hLAT4, model) and xAAT Phe transporters (xB0AT1, model; xLAT4, model; and xTAT1, model). n = 6–8 oocytes each experiment for 4 independent experiments. For panels (B,D), 95% confidence limits for predicted transporter activities are shown (dotted lines) bracketing the model predictions for each AAT activity.|