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

Abstract

We have investigated the functional role of Cl- in the human Na+/Cl-/γ-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 (K0.5) 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 GABAevoked currents and the unidirectional influx of 36Cl-, 22Na+, or [3H]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+ 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+:1C1-:1GABA per transport cycle. Activation of forward Na+/Cl-/ GABA transport evoked 36Cl- 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, C1- 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.