Rapid substrate-induced charge movements of the GABA transporter GAT1.
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.
Bicho
Ana
A
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Grewer
Christof
C
D000818:Animals; D001692:Biological Transport; D001703:Biophysics / Q000379:methods*; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D002712:Chlorides / Q000737:chemistry; D002713:Chlorine / Q000737:chemistry; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D007477:Ions; D007700:Kinetics; D008027:Light; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008956:Models, Chemical; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010782:Photolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012964:Sodium / Q000737:chemistry; D013997:Time Factors
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D007477:Ions; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C493413:SLC6A1 protein, human; C493414:Slc6a1 protein, mouse; D002713:Chlorine; D012964:Sodium