Cation and voltage dependence of rat kidney electrogenic Na(+)-HCO(-)(3) cotransporter, rkNBC, expressed in oocytes

Abstract

Recently, we reported the cloning and expression of the rat renal electrogenic Na(+)-HCO(-)(3) 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) HCO(-)(3) dependent, and 4) inhibited by stilbenes such as DIDS. The apparent HCO(-)(3):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 HCO(-)(3)-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(+) ( approximately 30 mM [Na(+)](o)) is voltage and HCO(-)(3) 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(+)-HCO(-)(3) 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 [HCO(-)(3)] 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 [HCO(-)(3)](o). rkNBC stoichiometry is 2 HCO(-)(3):1 Na(+) over this entire Na(+) range at both HCO(-)(3) 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 HCO(-)(3):1 Na(+) thought necessary for HCO(-)(3) reabsorption by the renal proximal tubule, a control mechanism or signal that alters its in vivo function is hypothesized.