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, respectively) expressed in Xenopus laevis oocytes, Substrate-evoked steady-state 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 movements that manifested as pre-steady-state current transients. Removal of Cl- shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K-0.5) for Cl- of 21 and 115 mM for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-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 Naf in the presence or absence of Cl-. Thus, the GAT1 stoichiometry was 2Na(+):1GABA, However, Cl- was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl- ion, suggesting unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activation of forward Na+/Cl-/GABA transport evoked Cl-36(-) efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl-/Cl- exchange mechanism during the GAT1 transport cycle. In contrast, Cl- was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl- modulates the kinetics of cotransport by altering Na+ affinity, but does not contribute to net charge transported per transport cycle, We conclude that Cl- dependence per se is not a useful criterion to classify Na+ cotransporters.