A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2

Abstract

Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl−. Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na+, and Cl− in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl− on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstrate ions and substrate such that their forward transport mode was maintained even under conditions of elevated intracellular Na+ or Cl−. The most important differences in the transport cycle of GlyT1 and GlyT2 arose from the kinetics of charge movement and the resulting voltage-dependent rate-limiting reactions: the kinetics of GlyT1 were governed by transition of the substrate-bound transporter from outward- to inward-facing conformations, whereas the kinetics of GlyT2 were governed by Na+ binding (or a related conformational change). Kinetic modeling showed that the kinetics of GlyT1 are ideally suited for supplying the extracellular glycine levels required for NMDA receptor activation.

Kinetic Models of Secondary Active Transporters

“…We tested this model by running simulations for three different transport stoichiometries (i.e., Na + /S, 2Na + /S and Na + /Cl − /S in the panels C, D and E, respectively, of Figure 1), and by comparing the resulting concentrations with the calculated values from the thermodynamic equation in Figure 1A. We stress that in these calculations (according to the reaction schemes outlined in panels C, D and E of Figure 1), we assume a hypothetical transporter: In Figure 1 and all subsequent figures, we chose to use rate constants, which describe the GlyT1 [16], but it is obvious that the stoichiometries were varied to an extent, which is not recapitulated by the experimental data for GlyT1.…”

“…The equation for the analytical solution can be readily derived in simple schemes like those shown in Figure 2A or Figure 2E, but this becomes substantially more challenging in complex models. However, these complex models, which encompass many additional loops, are required to explain experimental observations [16,19]. Thus, for more complex models, the simulations rather than the analytical solutions are the more practical approach to assess the effect of slippage on the intra- and extracellular substrate concentrations at the steady state.…”

“…Electrophysiological data are available for a large collection of sodium-dependent SLC6 transporters [16,19,21,22,23,24]. Based on these data, Na + /substrate coupling in SLC6 transporters appears to be quite efficient.…”

“…In this context, it is worth noting that for every SLC6 transporter, where this was explicitly explored, the binding of co-substrates and substrate was found to be cooperative [15,16,39,40]: Co-substrates and substrate bound per se with lower affinity than upon ternary complex formation (i.e., transporter, co-substrates and substrate). An important consequence of this has been frequently overlooked: Cooperativity, which implies that co-substrate and substrate bind in a random order, because a sequential binding scheme cannot account for cooperative co-substrate and substrate binding.…”

An Electrophysiological Approach to Measure Changes in the Membrane Surface Potential in Real Time

“…It was reported that GlyT2 but not GlyT1 bound extracellular Na + in a voltage-dependent manner. Voltage jumps from negative (i.e., −60 mV) to more positive potentials, resulted in outwardly directed transient currents, which were presumably carried by dissociating Na + ions ( 20 , 21 ). Our data suggest that the voltage at the membrane settles faster than Na + can unbind.…”