Cotransporters Cloned from Salamander and Rat Kidney

We studied the extracellular [HCO ] dependence of two renal clones of the electrogenic Na/HCO 3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential ( D 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 ( D I) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCO / CO 2 (0.33–99 mM HCO , pH o 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)sensitive and Na 1 -dependent hyperpolarization (or outward current). In D V m experiments, the apparent K m for HCO of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K m for HCO of rkNBC was less (6.5 mM). Because it has been reported that SO /HSO stimulates Na/ HCO 3 cotransport in renal membrane vesicles (a result that supports the existence of a CO binding site with which SO interacts), we examined the effect of SO /HSO on rkNBC. In voltage-clamp studies, we found that neither 33 mM SO nor 33 mM SO /HSO substantially affects the apparent K m for HCO . We also used microelectrodes to monitor intracellular pH (pH i ) while exposing rkNBC-expressing oocytes to 3.3 mM HCO /0.5% CO 2 . We found that SO /HSO did not significantly affect the DIDS-sensitive component of the pH i recovery from the initial CO 2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO 2 ] o , pH o , and [CO ] o at a fixed [HCO ] o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [CO ] 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 SO , HSO , or CO . key words: Xenopus oocytes • intracellular pH • extracellular pH • sulfite • carbonate 3 2