Rapid Substrate-Induced Charge Movements of the GABA Transporter GAT1

Abstract

The GABA transporter GAT1 removes the neurotransmitter GABA from the synaptic cleft by coupling of GABA uptake to the co-transport of two sodium ions and one chloride ion. The aim of this work was to investigate the individual reaction steps of GAT1 after a GABA concentration jump. GAT1 was transiently expressed in HEK293 cells and its pre-steady-state kinetics were studied by combining the patch-clamp technique with the laser-pulse photolysis of caged GABA, which allowed us to generate GABA concentration jumps within <100 micros. Recordings of transport currents generated by GAT1, both in forward and exchange transport modes, showed multiple charge movements that can be separated along the time axis. The individual reactions associated with these charge movements differ from the well-characterized electrogenic "sodium-occlusion" reaction by GAT1. One of the observed electrogenic reactions is shown to be associated with the GABA-translocating half-cycle of the transporter, in contradiction to previous studies that showed no charge movements associated with these reactions. Interestingly, reactions of the GABA-bound transporter were not affected by the absence of extracellular chloride, suggesting that Cl- may not be co-translocated with GABA. Based on the results, a new alternating access sequential-binding model is proposed for GAT1's transport cycle that describes the results presented here and those by others.

Turnover Rate of the γ-Aminobutyric Acid Transporter GAT1

“…Substituting these values in equation 7 yields a GAT1 unitary turnover rate of 15 ± 2 s −1 at 21°C and −50 mV. This estimate of the GAT1 turnover rate is higher than that reported in most previous investigations (Radian et al, 1986;Mager et al, 1993;Forlani et al, 2001b;Fesce et al, 2002) but closer to that reported in a recent study utilizing rapid concentration jumps for rat GAT1 (13 s −1 at −40 mV and room temperature; Bicho & Grewer, 2005).…”

“…The above kinetics data suggest that at standard Na + (100 mM) and Cl − (106 mM) concentrations used in the bathing medium of X. laevis oocytes, the GAT1 GABA-evoked current ( ) does not represent the maximum macroscopic current (i.e., I max ) (see also Bicho & Grewer, 2005). Thus, we examined whether a supersaturating GABA concentration could drive transport to its maximum velocity (even at subsaturating Na + and Cl − concentrations), as would be predicted by an ordered kinetic scheme in which GABA is the last cosubstrate to bind to the transporter from the extracellular side (see Parent et al, 1992;Eskandari et al, 1997;Hilgemann & Lu, 1999;Forster et al, 2002;Sacher et al, 2002;Whitlow et al, 2003;Karakossian et al, 2005).…”

“…Although the charge moved (Q NaCl ) is directly related to the total number of functional transporters in the plasma membrane (Zampighi et al, 1995;Chandy et al, 1997;Eskandari et al, 2000), the number of transporters obtained from Q NaCl relies on the electrophysiological estimate of zδ, as determined from a fit of the charge-voltage relationship with a single Boltzmann function (see Fig. 1b and equation 3) (Loo et al, 1993;Mager et al, 1993;Wang et al, 2003;Bicho & Grewer, 2005). Many electrogenic Na + -coupled transporters exhibit a value of unity for zδ, although values lower and higher than unity have also been reported (Loo et al, 1993;Mager et al, 1993;Wadiche et al, 1995;Eskandari et al, 1997;Hazama et al, 1997;Lu & Hilgemann, 1999b;Forster et al, 2002;Sacher et al, 2002;Karakossian et al, 2005).…”

Pre-steady-State Currents in Neutral Amino Acid Transporters Induced by Photolysis of a New Caged Alanine Derivative

“…Typical laser energies, measured at the emitting end of the optical fiber using an energy meter (Gentec), were in the range of 50-400 mJ/cm 2 . A standard alanine concentration of 1 mM was applied to the cell by rapid solution exchange before and after laser-pulse photolysis to estimate the concentration of photolytically released alanine and to test the cell for damage by the laser pulse, as described previously (18,21,26). Briefly, photolysis-induced steady-state currents (after decay of the transient components) at subsaturating alanine concentrations were compared with the steady-state current evoked by 1 mM free alanine and calibrated using an appropriate alanine dose-response curve for each transporter, as illustrated for ASTC2 in Figure 4A-C. Alanine dose-response curves were obtained by applying alanine at various concentrations to the transporter-expressing cells with a rapid solution exchange device and measuring the alanine-evoked steady-state current at each concentration.…”

“…A second method is based on applying concentration jumps of the transported substrate to the carrier while keeping the membrane potential constant, which also leads to relaxation to a new steady state. This method has been used with glutamate (12,17) and GABA (γ-aminobutyric acid) transporters (18) and has yielded a wealth of new information regarding the transport mechanism. However, rapid reactions of these secondary transporters were found to take place on the sub-millisecond to millisecond time scale (14), which makes substrate concentration jumps difficult to achieve with rapid mixing techniques.…”