The Renal Electrogenic Na⁺:HCO₃⁻ Cotransporter

The electrogenic Na⁺:HCO₃⁻ cotransporter (symporter) is the major transporter for HCO₃⁻ reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na⁺ reabsorption. We expression-cloned the salamander renal electrogenic Na⁺:Bicarbonate Cotransporter (NBC) in Xenopus laevis oocytes. After injecting poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectrodes to monitor membrane potential (V_m) and intracellular pH (pHi). All solutions contained ouabain to block the Na⁺/K⁺ pump (P-ATPase). After applying 1.5 % CO₂/10 mmol l⁻¹ HCO₃⁻ (pH 7.5) and allowing pHi to stabilize from the CO₂-induced acidification, we removed Na⁺. In native oocytes or water-injected controls, removing Na⁺ hyperpolarized the cell by −5 mV and had no effect on pHi. In oocytes injected with poly(A)+ RNA, removing Na⁺ transiently depolarized the cell by −10 mV and caused pHi to decrease; both effects were blocked by 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS) and required HCO₃⁻. We enriched the signal by electrophoretic fractionation of the poly(A)+ RNA, and constructed a size-selected cDNA library in pSPORT1 using the optimal fraction. Screening the Ambystoma library yielded a single clone (aNBC). Expression was first obvious 3 days after injection of NBC cRNA. Adding CO₂/HCO₃⁻ induced a large (>50 mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Subsequent Na⁺ removal depolarized the cell by more than 40 mV and decreased pHi. aNBC is a full-length clone with a start Met and a poly(A)+ tail; it encodes a protein with 1025 amino acids and several putative membrane-spanning domains. aNBC is the first member of a new family of Na⁺-linked HCO₃⁻ transporters. We used aNBC to screen a rat kidney cDNA library, and identified a full-length cDNA clone (rNBC) that encodes a protein of 1035 amino acids. rNBC is 86 % identical to aNBC and can be functionally expressed in oocytes.