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.

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.

1. Boron WF, and EL Boulpaep. Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport. J Gen Physiol 81: 53-94, 1983.
2. Romero MF, and W.F. Boron. Electrogenic Na/HCO3 cotransporters: Expression cloning and physiology. Ann. Rev. Physiol 61: in press, 1999.
3. Romero MF, P Fong, UV Berger, MA Hediger, and WF Boron. Cloning and functional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney. Am J Physiol 274: F425-32, 1998.
4. Romero MF, MA Hediger, EL Boulpaep, and WF Boron. Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter. Nature 387: 409-13, 1997.
5. Schmitt BM, Biemesderfer, D., Boulpaep, E.L., Romero, M.F., and Boron, W.F. Immunolocalization of the Electrogenic Na+/ HCO3- Cotransporter in Mammalian and Amphibian Kidney. Am. J. Physiol. in press, 1998.
6. Sciortino CM, and Romero, M.F. Na+ and voltage dependence of the rat kidney electrogenic Na/HCO3 cotransporter (rkNBC) expressed in Xenopus oocytes. J. Am. Soc. Nephrol. 9: 12A, 1998.