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 electrogenic Na⁺-HCO₃/^- cotransporter (rkNBC) in Xenopus oocytes [M. F. Romero, P. Fong, U. V. Berger, M. A. Hediger, and W. F. Boron. Am. J. Physiol. 274 (Renal Physiol. 43): F425-F432, 1998]. Thus far, all NBC cDNAs are at least 95% homologous. Additionally, when expressed in oocytes the NBCs are 1) electrogenic, 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 kidney to 2:1 in pancreas and brain to 1:1 in the heart. This study investigates the cation and voltage dependence of rkNBC expressed in Xenopus oocytes to better understand NBC's apparent tissue- specific physiology. Using two-electrode voltage clamp, we studied the cation specificity, Na⁺ dependence, and the current-voltage (I-V) profile of rkNBC. These experiments indicate that 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⁺ (~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 response is approximately linear; both inward and outward Na⁺-HCO₃/^- cotransport are observed. In contrast, only outward cotransport occurs at low [Na⁺](o) (<1 mM [Na⁺](o)). All rkNBC currents are inhibited by extracellular application of DIDS, independent of voltage and [Na⁺](o). Using ion-selective microelectrodes, we monitored intracellular pH and Na⁺ activity. We then calculated intracellular [HCO₃/^-] and, with the observed reversal potentials, calculated 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 Na⁺ over this entire Na⁺ range at both HCO₃/^- concentrations. Our results indicate that rkNBC is highly selective for Na⁺, with transport direction and magnitude 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 tubule, a control mechanism or signal that alters its in vivo function is hypothesized.