Publications

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

  Sciortino C.M., Romero M.F.

  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 le ... >> More

  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. << Less

  Am J Physiol 277:F611-23(1999) [PubMed] [EuropePMC]

• Functional characterization of human NBC4 as an electrogenic Na+-HCO cotransporter (NBCe2).

  Virkki L.V., Wilson D.A., Vaughan-Jones R.D., Boron W.F.

  We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own ... >> More

  We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pH(i)) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO- and Na+-dependent currents. When we voltage clamped V(m) far from NBC4c's estimated reversal potential (E(rev)), the pH(i) recovery rate increased substantially. Both the currents and pH(i) recovery were blocked by 200 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated the transporter's HCO:Na+ stoichiometry by measuring E(rev) at different extracellular Na+ concentration ([Na+]o) values. A plot of E(rev) against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E(rev) values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC. << Less

  Am. J. Physiol. 282:C1278-C1289(2002) [PubMed] [EuropePMC]

• The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO(3)(-):1 Na(+).

  Gross E., Abuladze N., Pushkin A., Kurtz I., Cotton C.U.

  The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the revers ... >> More

  The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane. << Less

  J Physiol 531:375-382(2001) [PubMed] [EuropePMC]