Abstract
SLC6A14 (solute carrier family 6 member 14) is an amino acid transporter, driven by Na+ and Cl− co-transport, whose structure, function, and molecular and kinetic mechanism have not been well characterized. Its broad substrate selectivity, including neutral and cationic amino acids, differentiates it from other SLC6 family members, and its proposed involvement in nutrient transport in several cancers suggest that it could become an important drug target. In the present study, we investigated SLC6A14 function and its kinetic mechanism after expression in human embryonic kidney (HEK293) cells, including substrate specificity and voltage dependence under various ionic conditions. We applied rapid solution exchange, voltage jumps, and laser photolysis of caged alanine, allowing sub-millisecond temporal resolution, to study SLC6A14 steady state and pre-steady state kinetics. The results highlight the broad substrate specificity and suggest that extracellular chloride enhances substrate transport but is not required for transport. As in other SLC6 family members, Na+ binding to the substrate-free transporter (or conformational changes associated with it) is electrogenic and is likely rate limiting for transporter turnover. Transient current decaying with a time constant of <1ms is also observed after rapid amino acid application, both in forward transport and homoexchange modes, indicating a slightly electrogenic, but fast and not rate-limiting substrate translocation step. Our results, which are consistent with kinetic modeling, suggest rapid transporter turnover rate and substrate translocation with faster kinetics compared with other SLC6 family members. Together, these results provided novel information on the SLC6A14 transport cycle and mechanism, expanding our understanding of SLC6A14 function.
Highlights
Plasma membrane amino acid transporters move amino acids across the lipophilic membrane bilayer into the cell (Zafra and Gimenez, 1986; Reyes et al, 2009; Wang et al, 2018), providing nutrients for many essential biological processes, such as protein and nucleotide synthesis, mammalian target of rapamycin signaling, and cell metabolism
The objective of this work was to determine the functional properties of SLC6A14 by electrophysiology in a cell system that allows control of the ionic composition of solutions on both sides of the membrane
To obtain the details of kinetic properties, we selected HEK293 cells to transiently over-express SLC6A14 cDNA and voltage clamped the cells in the whole-cell recording mode
Summary
Plasma membrane amino acid transporters move amino acids across the lipophilic membrane bilayer into the cell (Zafra and Gimenez, 1986; Reyes et al, 2009; Wang et al, 2018), providing nutrients for many essential biological processes, such as protein and nucleotide synthesis, mammalian target of rapamycin (mTOR) signaling, and cell metabolism. Amino acid transporter SLC6A14 belongs to the Solute Carrier 6 (SLC6) family, which consists of 20 membrane transporters in the human genome. It is known as ATB0,+ derived from transport selectivity for neutral amino acids, denoted by “0” and cationic amino acids denoted by “+.”. SLC6A14 is a unidirectional transporter, in which amino acid transport is coupled to Na+ and Cl− co-transport, with broad substrate selectivity (Sloan and Mager, 1999; Ugawa et al, 2001; Le Guellec et al, 2021), including all neutral and cationic amino acids It was identified as a β-alanine carrier (Bhutia et al, 2015). Amino acid transporter subfamily (II) comprised nutrient amino acid transporters SLC6A15-20 (Pramod et al, 2013; Rudnick et al, 2014; Coothankandaswamy et al, 2016)
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