Ionic mechanism of Na+-HCO3− cotransport in rabbit renal basolateral membrane vesicles

The exit of HCO3− across the basolateral membrane of the proximal tubule cell occurs via the electrogenic cotransport of 3 eq of base per Na+. We have used basolateral membrane vesicles isolated from rabbit renal cortex to identify the ionic species transported via this pathway. Media of varying pH and pCO2 were employed to evaluate the independent effects of HCO3− and CO32− on 22Na transport. Na+ uptake was stimulated when [CO32−] was increased at constant [HCO3−], indicating the existence of a transport site for CO32−. In the presence of HCO3−, Na+ influx was stimulated more than 3-fold by an inward SO32− gradient. SO32−-stimulated Na+ influx was stilbene-sensitive, confirming that it occurs via the Na+-HCO3− cotransport system. Na+-SO32− cotransport was demonstrated and found to have a 1:1 stoichiometry. Increasing [CO32−] at constant [HCO3−] reduced the stimulation of Na+ influx by SO32−, suggesting competition between SO32− and CO32− at a common divalent anion site. Additional divalent anions that were tested, such as SO42−, oxalate2-, and HPO4(2-), did not interact at this site. SO32− stimulation of Na+ influx was absolutely HCO3−(-)dependent and was increased as a function of [HCO3−], indicating the presence of a separate HCO3− site. Lastly, we tested whether Na+ interacts via ion pair formation with CO32− or binds to a distinct site. Na+, which has lower affinity than Li+ for ion pair formation with CO32−, was found to have > 5-fold higher affinity than Li+ for the Na+-HCO3− cotransport system. Moreover, when its inhibition was studied as a function of [Na+], harmaline was found to be a competitive inhibitor of Na+ influx, indicating the existence of a distinct cation site. Our data are compatible with a model in which base transport across the basolateral membrane of the proximal tubule cell takes place via 1:1:1 cotransport of CO32−, HCO3−, and Na+ on distinct sites.