Cation and voltage dependence of rat kidney electrogenic Na+-HCO3- cotransporter, rkNBC, expressed in oocytes

Recently, we reported the cloning and expression of the rat renal electroge nic Na+-HCO3- cotransporter (rkNBC) in Xenopus oocytes [M. F. Romero, P. Fo ng, U. V. Berger, M. A. Hediger, and W. F. Boron. Am. J. Physiol. 274 (Rena l Physiol. 43): F425-F432, 1998]. Thus far, all NBC cDNAs are at least 95% homologous. Additionally, when expressed in oocytes the NBCs are 1) electro genic, 2) Na+ dependent, 3) HCO3- dependent, and 4) inhibited by stilbenes such as DIDS. The apparent HCO3-:Na+ coupling ratio ranges from 3:1 in kidn ey to 2:1 in pancreas and brain to 1:1 in the heart. This study investigate s the cation and voltage dependence of rkNBC expressed in Xenopus oocytes t o better understand NBC's apparent tissue-specific physiology. Using two-el ectrode voltage clamp, we studied the cation specificity, Na+ dependence, a nd the current-voltage (I-V) profile of rkNBC. These experiments indicate t hat K+ and choline do not stimulate HCO3--sensitive currents via rkNBC, and Li+ elicits only 3 +/- 2% of the total Na+ current. The Na+ dose response studies show that the apparent affinity of rkNBC for extracellular Na+ (sim ilar to 30 mM [Na+](o)) is voltage and HCO3- independent, whereas the rkNBC I-V relationship is Na+ dependent. At [Na+](o) v(max) (96 mM), the I-V res ponse is approximately linear; both inward and outward Na+-HCO3- cotranspor t are observed. In contrast, only outward cotransport occurs at low [Na+](o ) (<1 mM [Na+](o)). All rkNBC currents are inhibited by extracellular appli cation of DIDS, independent of voltage and [Na+](o). Using ion-selective mi croelectrodes, we monitored intracellular pH and Na+ activity. We then calc ulated intracellular [HCO3-] and, with the observed reversal potentials, ca lculated the stoichiometry of rkNBC over a range of [Na+](o) values from 10 to 96 mM at 10 and 33 mM [HCO3-](o). rkNBC stoichiometry is 2 HCO3-:1 Naover this entire Na+ range at both HCO3- concentrations. Our results indica te that rkNBC is highly selective for Na+, with transport direction and mag nitude sensitive to [Na+](o) as well as membrane potential. Since the rkNBC protein alone in oocytes exhibits a stoichiometry of less than the 3 HCO3- :1 Na+ thought necessary for HCO3- reabsorption by the renal proximal tubul e, a control mechanism or signal that alters its in vivo function is hypoth esized.