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

Shi, Yueyue; Wang, Jiali; Ndaru, Elias; Grewer, Christof

doi:10.3389/fphys.2021.777050

Cited by 8 publications

(8 citation statements)

References 60 publications

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“…The kinetics of reaction steps associated with ASCT2 substrate translocation were previously studied using rapid alanine application with laser photolysis of caged-alanine [ 5 , 8 , 34 , 42 ]. In this previous study, transient currents induced by alanine application decayed with a time constant of 3 ms, providing information about the electrogenic steps in the translocation equilibrium [ 20 , 21 , 22 , 43 , 44 , 45 ].…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid substrate was applied through a theta capillary glass tubing (TG200-4, OD = 2.00 mm, ID = 1.40 mm. Warner Instruments, LLC, MA, USA), with the tip of the theta tubing pulled to a diameter of 350 μm and positioned at 0.5 mm to the cell [ 34 ]. For paired-pulse experiments, currents were recorded with varying interval time after removal of amino acid, starting at 10 ms.…”

Section: Methodsmentioning

confidence: 99%

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Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

2022

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Neutral amino acid transporters ASCT1 and ASCT2 are two SLC1 (solute carrier 1) family subtypes, which are specific for neutral amino acids. The other members of the SLC1 family are acidic amino acid transporters (EAATs 1–5). While the functional similarities and differences between the EAATs have been well studied, less is known about how the subtypes ASCT1 and 2 differ in kinetics and function. Here, by performing comprehensive electrophysiological analysis, we identified similarities and differences between these subtypes, as well as novel functional properties, such as apparent substrate affinities of the inward-facing conformation (in the range of 70 μM for L-serine as the substrate). Key findings were: ASCT1 has a higher apparent affinity for Na+, as well as a larger [Na+] dependence of substrate affinity compared to ASCT2. However, the general sequential Na+/substrate binding mechanism with at least one Na+ binding first, followed by amino acid substrate, followed by at least one more Na+ ion, appears to be conserved between the two subtypes. In addition, the first Na+ binding step, presumably to the Na3 site, occurs with high apparent affinity (<1 mM) in both transporters. In addition, ASCT1 and 2 show different substrate selectivities, where ASCT1 does not respond to extracellular glutamine. Finally, in both transporters, we measured rapid, capacitive charge movements upon application and removal of amino acid, due to rearrangement of the translocation equilibrium. This charge movement decays rapidly, with a time constant of 4–5 ms and recovers with a time constant in the 15 ms range after substrate removal. This places a lower limit on the turnover rate of amino acid exchange by these two transporters of 60–80 s−1.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

2022

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“…SLC6 transporters share a common mechanism of substrate translocation, but there are substantial variations in the partial reactions of the transport cycle: turnover numbers can, for instance, vary by more than an order of magnitude from less than 1 s −1 in the norepinephrine transporter NET/SLC6A2 ( Bhat et al, 2021b ) to 50 s −1 in the amino acid transporter SLC6A14 ( Shi et al, 2021 ) Similarly, even the very closely related monoamine transporters - i.e. the transporters for dopamine, serotonin and norepinephrine - differ in their ability to harvest the potassium gradient or the membrane potential ( Bhat et al, 2021b ).…”

Section: Discussionmentioning

confidence: 99%

Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1

et al. 2022

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Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determined the kinetics of CRT-1 in real time by relying on electrophysiological recordings of transport-associated currents. Our analysis revealed that CRT-1 harvested the concentration gradient of NaCl and the membrane potential but not the potassium gradient to achieve a very high concentrative power. We investigated the mechanistic basis for the ability of CRT-1 to maintain the forward cycling mode in spite of high intracellular concentrations of creatine: this is achieved by cooperative binding of substrate and co-substrate ions, which, under physiological ion conditions, results in a very pronounced (i.e. about 500-fold) drop in the affinity of creatine to the inward-facing state of CRT-1. Kinetic estimates were integrated into a mathematical model of the transport cycle of CRT-1, which faithfully reproduced all experimental data. We interrogated the kinetic model to examine the most plausible mechanistic basis of cooperativity: based on this systematic exploration, we conclude that destabilization of binary rather than ternary complexes is necessary for CRT-1 to maintain the observed cytosolic creatine concentrations. Our model also provides a plausible explanation why neurons, heart and skeletal muscle cells must express a creatine releasing transporter to achieve rapid equilibration of the intracellular creatine pool.

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“…It is important to note, however, that there is not any relation between the K M value and the V max value, which a priori dictates that these two parameters must move in the same direction. We examined the relation between turnover rates and K M in the SLC6 family, because turnover rates have been determined with high precision by electrophysiological recordings (Bicho and Grewer, 2005;Erdem et al, 2019;Bhat et al, 2021;Shi et al, 2021) and the individual steps of the transport cycle have been analyzed in detail. In addition, the K M values for cognate substrate span more than two orders of magnitude.…”

Section: Discussionmentioning

confidence: 99%

Optimizing the Substrate Uptake Rate of Solute Carriers

et al. 2022

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The diversity in solute carriers arose from evolutionary pressure. Here, we surmised that the adaptive search for optimizing the rate of substrate translocation was also shaped by the ambient extracellular and intracellular concentrations of substrate and co-substrate(s). We explored possible solutions by employing kinetic models, which were based on analytical expressions of the substrate uptake rate, that is, as a function of the microscopic rate constants used to parameterize the transport cycle. We obtained the defining terms for five reaction schemes with identical transport stoichiometry (i.e., Na+: substrate = 2:1). We then utilized an optimization algorithm to find the set of numeric values for the microscopic rate constants, which provided the largest value for the substrate uptake rate: The same optimized rate was achieved by different sets of numerical values for the microscopic rate constants. An in-depth analysis of these sets provided the following insights: (i) In the presence of a low extracellular substrate concentration, a transporter can only cycle at a high rate, if it has low values for both, the Michaelis–Menten constant (KM) for substrate and the maximal substrate uptake rate (Vmax). (ii) The opposite is true for a transporter operating at high extracellular substrate concentrations. (iii) Random order of substrate and co-substrate binding is superior to sequential order, if a transporter is to maintain a high rate of substrate uptake in the presence of accumulating intracellular substrate. Our kinetic models provide a framework to understand how and why the transport cycles of closely related transporters differ.

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

Cited by 8 publications

References 60 publications

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1

Optimizing the Substrate Uptake Rate of Solute Carriers

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