Expression cloning of the renal electrogenic Na/HCOs cotransporter (NBC-1) from ambystoma hgrinium

The electrogenic Na/HCOscotransporter is the major route for HCOà reabsorption, and also contributes to Na+ reabsorption, across the basolateral membrane of the renal proximal tubule (PT). Boron and Boulpaep [J Gen Physiol 81:53,1983) first described and characterized this (transporter in the PT of the salamander Ambystoma tigrinium. and others have subsequently identified it in mammalian PT's and a variety of other cells. We have used the Xenopus cocyte expression cloning strategy [Hediger et al. Nature 330:339,1987] to clone the renal electrogenic da Bicarbonate Eotransporter (N9C-1). After injection of polyA-, fractionated-, or c-RNA, cocytes were analyzed using microelectrodes to measure membrane potential {Vm)and intracellular pH (pHj). Our assay was to apply 1.5% C(V 10 mM HCOa(pH 7.5), to wait for pH to stabilize (C02-pHj i), and then to remove Na+. All solutions contained 100 u.M ouabain to block the oocyte's Na⁺/K⁺ ATPase. In native or water-injected controls, Na+ removal did NOT alter pH, and hyperpolarized the cell by -6mV. In oocytes infected polyA- or fractionated-RNA removing Na+ reveals a transient depolarization !-10mVj and a pH, i, both of which are blocked by 200 U.M DIDS and are absolutely HCOjdependent. This expression pattern occurred between days 7-13 post RNA injection. We enriched the signal by electrophoretic fracttonation of the potyA-RNA and used the optimal fraction to construct a size-selected cDNA library in pSPORTt. Screening the library yielded a single clone, NBC-1, that manifested the cotransporter's expression pattern. Expression of NBC-1 is first obvious 3 days post injection. Adding CLVHCOs induces a large (>50 mV) and rapkj hyperpolarization, followed by a partial relaxation as pH, stabilizes. Subsequent Na+ removal depolarizes the cell by >40mV and -l's pH|. Readdition of Na⁺ reverses these effects. Lowering HC0.3(10-2mM) at constant PCO?I mimics the effect of Na+ removal. 200 (iM DIDS completely blocks the i's in Vm and pH(. NBC-1 is a 4 kb cDNA insert that encodes a large protein with several putative membrane-spanning domains. Comparing the NBC-1 sequence to the GenBank database revealed no substantial homology to existing proteins, indicating that NBC-1 is the first member of a new family of Na+-linked HCOâ transporters. (NIH grants DK30344, DK17433. 8 DK43171, the National KKiney Foundation, and NRSA DK09342).