Extracellular HCO₃/^- dependence of electrogenic Na/HCO₃ cotransporters cloned from salamander and rat kidney

We studied the extracellular [HCO₃/^-] dependence of two renal clones of the electrogenic Na/HCO₃ cotransporter (NBG) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔV(m)) elicited by the NBC cloned from the kidney of the salamander Arabystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO₃/^-/ CO₂ (0.33-99 mM HCO₃/^-, pH(o) 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)sensitive and Na⁺- dependent hyperpolarization (or outward current). In ΔV₃(m) experiments, the apparent K(m) for HCO₃/^- of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCO₃/^- of rkNBC was less (6.5 mM). Because it has been reported that SO₃/(=)/HSO₃/^- stimulates Na/ HCO₃ cotransport in renal membrane vesicles (a result that supports the existence of a CO₃/(=) binding site with which SO₃/(=) interacts), we examined the effect of SO₃/(=)/HSO₃/^- on rkNBC. In voltage-clamp studies, we found that neither 33 mM SO₄/(=) nor 33 mM SO₃/(=)/HSO₃/^- substantially affects the apparent K(m) for HCO₃/^-. We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCO₃/^-/0.5% CO₂. We found that SO₃/(=)/HSO₃/^- did not significantly affect the DIDS-sensitive component of the pH(i) recover), from the initial CO₂-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO₂](o), pH(o), and [CO₃/(=)](o) at a fixed [HCO₃/^-](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [CO₃/(=)](o). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SO₃/(=), HSO₃/^-, or CO₃/(=).