Invited Symposium: Na-H Exchangers and Intracellular pH Regulation
NOTE to the reader: We apologize for the apparent lack of details in this presentation. However, since our abstract represents new and previously unpublished results, providing those results in this Internet forum would constitute publication. Presenting these results in this forum with current copyright practices would prohibit us from publishing in peer review journals in which we would otherwise publish. Consequently, we will only provide text descriptions and general conclusions of our work.
The kidney is reabsorbs virtually all of HCO3 filtered by the glomerulus. About 90% of the filtered HCO3 is reabsorbed in the proximal tubule. An electrogenic Na/HCO3 cotransporter was shown to be the mechanism by which NaHCO3 is transported across the basolateral membrane of proximal tubule cells. This cotransporter was later cloned by expression in Xenopus oocytes from salamander kidney and by homology in the rat kidney, human brain, pancreas, and hear. The cloned electrogenic Na bicarbonate cotransporter (NBC) cDNA codes for either a 1035 (kidney) or 1079 amino acid protein with 10-12 putative transmembrane domains. Study of the basic properties of NBC expressed in oocytes has shown the protein to be (1) HCO3 dependent, (2) Na dependent, (3) electrogenic and (4) inhibitable by the stilbene compound DIDS. The present study looks at the cation and voltage dependence of the rat kidney clone (rkNBC) expressed in oocytes. We used two electrode voltage clamp and ion selective microelectrodes to determine what monovalent cations could stimulate HCO3 transport as well as the voltage range of rkNBC activity. In addition we describe the Na dependence of rkNBC. Finally, from the current voltage analysis of rkNBC and with the measured values of intracellular Na/HCO3 concentrations we also calculate the HCO3 : Na stoichiometry of transport through rkNBC.
Materials and Methods
Oocytes are removed and defolliculated as previously described. rkNBC cRNA was injected and DIDS inhibitable, hco3 stimulated responses studied 3-12 days after injection. Two-electrode voltage clamp is used to monitor currents elicited by altering the HCO3 and cation concentrations of the bath solution. Current voltage protocols (IV) are executed by stepping the membrane potential from the holding potential of -60 mV to test potential (-160 to 60 mV in 20 mV increments), and recording the current response.
Ion selective microelectrodes filled either with H+ or Na selective resins are used to monitor intracellular ion concentrations of oocytes during the experiments. These electrodes are determined by two point calibrated with slopes of at least -56 mV/decade. In addition Na electrodes, tested for selectivity over K with KCl, have at least a 10 times selectivity for Na.
To determine the cation selectivity of rkNBC oocytes, bath solutions are changed from non- HCO3 to 1.5% CO2/10 mM HCO3/pH 7.5 solutions in the presence of 96 mM cation (Na+, K+, Li+, or choline). IVs are measured in both solutions, and the HCO3 stimulated current taken as the difference between the HCO3 and non-HCO3 solution IV.
The Na-dependence of rkNBC expressing oocytes is tested by holding an oocytes potential at -60 mV and perfusing an oocyte for 10 min in 1.5% CO2/10 mM HCO3/pH 7.5 in 96 mM Na for 10 min. This is followed by short solution pulses to 0 na, followed by a test Na concentration and then to 96 mM Na. IVs are recorded at the maximal current at the holding potential resulting from each change of Na. IVs are recorded at the maximal current at the holding potential resulting from each change of Na.
In the presence of bath Na/HCO3, rkNBC elicits a voltage dependent current. At voltages more hyperpolarized than the reversal potential, only negative current is observed. At more positive voltages, only positive current is seen. This is representative of two directions of rkNBC cotransport. Positive currents indicate inward Na/HCO3 transport whereas negative currents represent outward Na/hco3 cotransport. The direction and magnitude depend on the cell's membrane potential. However, when no Na is present in the bath solution, only negative currents are observed. Likewise when Na is replaced with Li, K, or choline, a HCO3 stimulated current is not measured.
Using ion selective microelectrodes we measured the intracellular aNa and pH over the course of the Na dose experiments. Using the Henderson-Hasselbach equation we determine the intracellular Na and HCO3 concentrations when rkNBC reaches steady state in 1.5% CO2/10 mM HCO3/pH 7.5 to be ~ 9 mM and 5 mM respectively. Using these values, the recorded reversal potentials from the IV recordings and the extracellular Na and HCO3 concentrations, we determine the HCO3 : Na stoichiometry of rkNBC cotransport versus external Na concentration (Boron & Boulpaep, JGP 81:53 1983). We find that this stoichiometry is 2:1 at all Na concentrations from 10 to 96 mM.
Discussion and Conclusion
This study reports the voltage and cation dependence of the electrogenic Na/HCO3 cotransporter from the rat kidney (rkNBC). We find that this protein expressed in Xenopus oocytes is specific for Na over Li, K and choline. This potentially indicates a means by which NBC can function in various ionic environments and contain a high sensitivity for Na/HCO3 dependent transport.
The stoichiometry of the electrogenic Na/HCO3 cotransporter in the renal proximal tubule should be at least 2.5 : 1 in order for HCO3 to move out of the proximal tubule cell. However, we find in our experiments that rkNBC expressed in oocytes has a stoichiometry of 2:1. These results imply that some other mechanism is involved in modulating rkNBCs function. Such modulating "factors" could include an accessory protein or an environmental component not yet identified.
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|Sciortino, C.M.; Romero, M.F.; (1998). Cation and Voltage Dependence of the Rat Kidney Electrogenic Na/HCO3 Cotransporter (rkNBC) Expressed in Xenopus Oocytes.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/fliegel/sciortino0797/index.html|
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