A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling

Abstract

Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xenopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.

Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii

“…These data suggested that TgApiAT6-1 may be a Lys transporter, although it could also mediate the uptake of other amino acids not detected under the transport conditions of the experiments, or not detected by GC-MS, such as Arg. Arg is readily converted into L-ornithine during sample preparation for GC-MS such that very little remains chemically unmodified [34,35].…”

“…To measure whether AA + accumulation occurs in TgApiAT6-1 and TgApiAT1-expressing oocytes, we used a targeted LC-MS/MS approach to measure intra-oocyte substrate concentrations while accounting for the endogenous metabolism of these substrates [35].…”

“…were incubated with substrate at concentrations, pH and temperatures indicated in figure legends. All incubations occurred in substrates solved in 1 × ND96 buffer (1.8 mM CaCl2, pH 7.3) as described previously [35]. For each condition, 12 oocytes were washed × 3 with 1 × ND96 solution not containing substrate under ambient conditions, before addition of 1 × ND96 containing substrate.…”

“…Sequant, Merck) as described previously [35]. The gradient started with 80% mobile phase B (Acetonitrile; 0.1% v/v Formic acid) and 20% mobile phase A (10mM ammonium formate; 0.1% v/v formic acid) at a flow rate of 300 µl/min, followed by a linear gradient to 20% mobile phase B over min.…”

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure–Function Relationships in Mammalian SLC6 Transporters

“…Furthermore, in this work, the presence of substrates normally not present in the cytoplasmatic environment, allowed us to develop an alternative method for investigating the actual translocation of the substrate inside the cell, we evaluated the amount of D-amino acids in the single oocyte by analyzing the cytoplasmic content by HPLC [ 120 ]. This method has only recently been implemented by the use of GS-MS allowing to measure in a determined and precise way the basal content of metabolite in oocytes and, in oocytes expressing different membrane transporters, the amount of transported substrate in a single cell after exposure to different medium [ 145 ]. Finally, it is important to underline how the transport of D-amino acids by the insect NATs could be of particular interest, given the recent approach that involves the use of insects for the treatment of organic waste for the production of molecules of nutraceutical, pharmacological and nutritional interest [ 146 , 147 ].…”