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 (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (Delta V-m) elicited by the NBC cloned from the kidney of the salamander Ambystoma 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 (Delta I) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO3-/CO2 (0.33-99 mM HCO3-, PHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)- sensitive and Na+-dependent hyperpolarization (or outward current). In Delta V-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) recovery 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=.