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Original Article

^a From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520.203-785-4951

walter.boron{at}yale.edu

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 (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 (I) 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 V_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⁼.

Key Words: Xenopus oocytes • intracellular pH • extracellular pH • sulfite • carbonate

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