Role of Cl- in electrogenic Na+-coupled cotransporters GAT1 and SGLT1

We have investigated the functional role of Cl- in the human Na+/Cl-/gamma -aminobutyric acid (GABA) and Na+/glucose cotransporters (GAT1 and SGLT1, r espectively) expressed in Xenopus laevis oocytes, Substrate-evoked steady-s tate inward currents were examined in the presence and absence of external Cl-. Replacement of Cl- by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na+ and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge mov ements that manifested as pre-steady-state current transients. Removal of C l- shifted the voltage dependence of charge movements to more negative pote ntials, with apparent affinity constants (K-0.5) for Cl- of 21 and 115 mM f or SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GA BA-evoked currents and the unidirectional influx of Cl-36(-), Na-22(+), or [H-3]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na f in the presence or absence of Cl-. Thus, the GAT1 stoichiometry was 2Na():1GABA, However, Cl- was transported by GAT1 because the inward movement o f 2 positive charges was accompanied by the influx of one Cl- ion, suggesti ng unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activa tion of forward Na+/Cl-/GABA transport evoked Cl-36(-) efflux and was block ed by the inhibitor SKF 89976A. These data suggest a Cl-/Cl- exchange mecha nism during the GAT1 transport cycle. In contrast, Cl- was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl- modulates the kinetics of cotra nsport by altering Na+ affinity, but does not contribute to net charge tran sported per transport cycle, We conclude that Cl- dependence per se is not a useful criterion to classify Na+ cotransporters.