Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation

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 i...

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Autores principales:	Yueyue Shi, Jiali Wang, Elias Ndaru, Christof Grewer
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	membrane transporter SLC6A14 ATB0,+ electrophysiology rapid kinetics laser-photolysis Physiology QP1-981
Acceso en línea:	https://doaj.org/article/ed931a4e9a9440dfb4383a7146aa37a1
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spelling	oai:doaj.org-article:ed931a4e9a9440dfb4383a7146aa37a12021-11-16T07:48:24ZPre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation1664-042X10.3389/fphys.2021.777050https://doaj.org/article/ed931a4e9a9440dfb4383a7146aa37a12021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fphys.2021.777050/fullhttps://doaj.org/toc/1664-042XSLC6A14 (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.Yueyue ShiJiali WangElias NdaruChristof GrewerFrontiers Media S.A.articlemembrane transporterSLC6A14ATB0,+electrophysiologyrapid kineticslaser-photolysisPhysiologyQP1-981ENFrontiers in Physiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	membrane transporter SLC6A14 ATB0,+ electrophysiology rapid kinetics laser-photolysis Physiology QP1-981
spellingShingle	membrane transporter SLC6A14 ATB0,+ electrophysiology rapid kinetics laser-photolysis Physiology QP1-981 Yueyue Shi Jiali Wang Elias Ndaru Christof Grewer Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation
description	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.
format	article
author	Yueyue Shi Jiali Wang Elias Ndaru Christof Grewer
author_facet	Yueyue Shi Jiali Wang Elias Ndaru Christof Grewer
author_sort	Yueyue Shi
title	Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation
title_short	Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation
title_full	Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation
title_fullStr	Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation
title_full_unstemmed	Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation
title_sort	pre-steady-state kinetic analysis of amino acid transporter slc6a14 reveals rapid turnover rate and substrate translocation
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/ed931a4e9a9440dfb4383a7146aa37a1
work_keys_str_mv	AT yueyueshi presteadystatekineticanalysisofaminoacidtransporterslc6a14revealsrapidturnoverrateandsubstratetranslocation AT jialiwang presteadystatekineticanalysisofaminoacidtransporterslc6a14revealsrapidturnoverrateandsubstratetranslocation AT eliasndaru presteadystatekineticanalysisofaminoacidtransporterslc6a14revealsrapidturnoverrateandsubstratetranslocation AT christofgrewer presteadystatekineticanalysisofaminoacidtransporterslc6a14revealsrapidturnoverrateandsubstratetranslocation
_version_	1718426640947609600

Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation

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