THERMODYNAMIC DETERMINATION OF THE NA- GLUCOSE COUPLING RATIO FOR THEHUMAN SGLT1 COTRANSPORTER( )

Phlorizin-sensitive currents mediated by a Na-glucose cotransporter we re measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA, Using a two-microelectrode voltage clamp technique, measured reversal potentials (V-r) at high external alpha- methylglucose (alpha MG) concentrations were linearly related to In[al pha MG](o), and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule, As [ alpha MG](o) decreased below 0.1 mM, V-r was no longer a linear functi on of In[alpha MG](o), in accordance with the suggested capacity of SG LT1 to carry Na in the absence of sugar (the ''Na leak''). A generaliz ed kinetic model for SGLT1 transport introduces a new parameter, K-c, which corresponds to the [alpha MG](o) at which the Na leak is equal i n magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of V-r as a function of In[alpha MG](o) could be fitted ove r the entire range of [alpha MG](o) if K-c is adjusted to 40 +/- 12 mu M. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absenc e of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong i nward rectification. The affinity of alpha MG for its internal site wa s low; the K-m was estimated to be between 25 and 50 mM, an order of m agnitude higher than that found for the extracellular site. Furthermor e, V-r determinations at varying alpha MG concentrations indicate a tr ansport stoichiometry of 2 Na ions per alpha MG molecule: the slope of V-r versus In[alpha MG](o) averaged 30.0 +/- 0.7 mV/decade (correspon ding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule ) whenever [alpha MG](o) was higher than 0.1 mM. These direct observat ions firmly establish that Na ions can utilize the SGLT1 protein to cr oss the membrane either alone or in a coupled manner with a stoichiome try of 2 Na ions per sugar molecule.