The Actual Ionic Nature of the Leak Current through the Na+/Glucose Cotransporter SGLT1

Abstract

Expression of the Na(+)/glucose cotransporter SGLT1 in Xenopus oocytes is characterized by a phlorizin-sensitive leak current (in the absence of glucose) that was originally called a "Na(+) leak" and represents some 5-10% of the maximal Na(+)/glucose cotransport current. We analyzed the ionic nature of the leak current using a human SGLT1 mutant (C292A) displaying a threefold larger leak current while keeping a reversal potential (V(R)) of approximately -15 mV as observed for wt SGLT1. V(R) showed only a modest negative shift when extracellular Na(+) concentration ([Na(+)](o)) was lowered and it was completely insensitive to changes in extracellular Cl(-). When extracellular pH (pH(o)) was decreased from 7.5 to 6.5 and 5.5, V(R) shifted by +15 and +40 mV, respectively, indicating that protons may be the main charge carrier at low pH(o) but other ions must be involved at pH(o) 7.5. In the presence of 15 mM [Na(+)](o) (pH(o) = 7.5), addition of 75 mM of either Na(+), Li(+), Cs(+), or K(+) generated similar increases in the leak current amplitude. This observation, which was confirmed with wt SGLT1, indicates a separate pathway for the leak current with respect to the cotransport current. This means that, contrary to previous beliefs, the leak current cannot be accounted for by the translocation of the Na-loaded and glucose-free cotransporter. Using chemical modification and different SGLT1 mutants, a relationship was found between the cationic leak current and the passive water permeability suggesting that water and cations may share a common pathway through the cotransporter.

An Externally Accessible Linker Region in the Sodium-Coupled Phosphate Transporter PiT-1 (SLC20A1) is Important for Transport Function

“…This electrogenic response might be evidence of an uncoupled leak current that is suppressed during the cotransport cycle. Analogous behavior has been reported for SLC34A1 proteins for which an uncoupled leak can be detected using the P i -transporter inhibitor phosphonoformic acid [46], the sodium-coupled glucose cotransporter SGLT1 for which pflorizin is known to block a protein-associated uncoupled leak [47,48] and other cation-coupled cotransporters. Presently there is no PiT-specific inhibitor available to investigate this phenomenon further in the SLC20 family.…”

Surprising Substrate Versatility in SLC5A6

“…For NIS and SGLT1, the Na ϩ leak currents reached ϳ35 and ϳ10% of the maximum Na ϩ / substrate cotransport currents, respectively, and they are attributed to the uncoupled flux of Na ϩ in the absence of substrate (12,(21)(22)(23). However, the molecular basis of the leak currents has recently been challenged by Longpre et al (24). The authors show that the transporter-associated cation leak current is directly proportional to the SGLT1-mediated passive water permeability, and they suggest that water and cations share a common pathway through the transporter.…”

The Structural Pathway for Water Permeation through Sodium-Glucose Cotransporters

“…Water permeability was evaluated by optical measurements on oocytes challenged with hypotonic media. Details of the procedure are given in previous publications dealing with SGLT1 and water transport (5,10,27). Briefly, the experimental chamber (volume of 0.07 ml) was specifically designed to enable rapid solution changes (solution flux of 1.2 ml/min), thus allowing measurement of rapid changes in oocyte volume.…”