Extracellular Hco₃⁻ Dependence of Electrogenic Na/Hco₃ Cotransporters Cloned from Salamander and Rat Kidney

We studied the extracellular [HCOabstract ₃ ⁻] 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 (ΔV_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 (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract ₃ ⁻/CO₂ (0.33–99 mM HCOabstract ₃⁻, pH_o 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na⁺-dependent hyperpolarization (or outward current). In ΔV_m experiments, the apparent K_m for HCOabstract ₃⁻ of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K_m for HCOabstract ₃⁻ of rkNBC was less (6.5 mM). Because it has been reported that SOabstract ₃⁼/HSO abstract ₃⁻ stimulates Na/HCO₃ cotransport in renal membrane vesicles (a result that supports the existence of a COabstract ₃⁼ binding site with which SOabstract ₃⁼ interacts), we examined the effect of SOabstract ₃⁼/HSO abstract ₃⁻ on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract ₄⁼ nor 33 mM SOabstract ₃ ⁼/HSOabstract ₃⁻ substantially affects the apparent K_m for HCO abstract ₃⁻. We also used microelectrodes to monitor intracellular pH (pH_i) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract ₃ ⁻/0.5% CO₂. We found that SO abstract ₃⁼/HSOabstract ₃ ⁻ 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 [COabstract ₃⁼]_o at a fixed [HCOabstract ₃⁻]_o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract ₃⁼]_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 SOabstract ₃ ⁼, HSOabstract ₃⁻, or COabstract ₃⁼.