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

Abstract

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.

Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells

“…5 B) after correction for the linear and unsaturable uptake process. Our Km values obtained using either NLuc luminescence or intracellular CT as readouts are lower but in close agreement to the range of published values (22–52 μM) 7 , 8 , 39 , 40 .

Figure 5 Comparison of creatine (CT) uptake kinetics attributed to CT transporter (CrT) derived from intracellular CT levels and AGAT-NLuc activity in response to extracellular CT levels (1–1,000 μM range).

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“…Fitting the dose response data to a two-phase decay curve resulted in two predicted inflection points at 0.02 mM and 2 mM. The first inflection point value is close to the published experimentally derived Km for CrT of 22–52 μM 7 , 8 , 39 , 40 , emphasizing that functional CrT is crucial for the intracellular response to extracellular CT exposure in concentrations less than 100 μM.

Figure 2 Change in AGAT-NLuc activity and intracellular creatine (CT) with increasing extracellular CT.

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Probing binding and occlusion of substrate in the human creatine transporter‐1 by computation and mutagenesis

“…A cysteine at the Y148 position would not be able to form a hydrogen bond with the carboxyl group of creatine, and so a critical link in the interaction chain would be lost, leading to transporter inactivation. This model is also consistent with experimental data that shows that CRT1 binds creatine and the co‐transported ions in a highly cooperative manner (Farr et al, 2022 ). Our computational data suggests this cooperativity could come, in part, from the essential interaction between creatine and Na + in the Na1 binding site.…”

“…This uphill transport is accomplished by CRT1, a member of the solute carrier 6 family (SLC6) (Guimbal & Kilimann, 1993 ). CRT1 utilizes the Na + /Cl − concentration gradient to actively transport one creatine molecule per one Cl − and two Na + ions (Farr et al, 2022 ; Peral et al, 2002 ). Like all of the membrane transporters, the creatine transporter (SLC6A8) cycles through a series of conformational states, including an extracellularly facing outward‐open (OO) state, a sealed occluded state, and an inwardly facing inward‐open state, in order to translocate creatine across the lipid bilayer (Drew & Boudker, 2016 ; Jardetzky, 1966 ).…”

Orphan lysosomal solute carrier MFSD1 facilitates highly selective dipeptide transport

“…2 B ). The rate of current deactivation upon removal of the substrate from the bath solution is a surrogate for the substrate turnover rate of a SLC ( 15 , 16 ). A monoexponential fit to the current decay (see Inset in Fig.…”