HCO 3 - reabsorption by the kidney One of the major functions of the renal proximal tubule is to reclaim HCO 3 - that has been filtered in the glomerulus. This reabsorption of HCO3 - from the proximal-tubule lumen to the blood helps to maintain an appropriately high [HCO 3 - ] in the blood and thus helps to stabilize blood pH. Failure to reabsorb sufficient HCO3 - would lead not only to the loss of HCO 3 - in the urine but also to the loss of Na + (which normally accompanies reabsorbed HCO 3 - ) and osmotically obligated water. Thus, the consequences of reduced Na + reabsorption would be metabolic acidosis and volume depletion. As shown in Fig. 1, HCO 3 - reabsorption by the proximal tubule is a multistep process. First, H + is secreted into the lumen by Na + /H + exchangers (antiporters) and presumably vacuolar-type H + pumps (V-type ATPases). Second, this H + titrates luminal HCO 3 - to CO 2 and H 2 O, a process accelerated by carbonic anhydrase IV (CA IV), which is tethered to the extracellular surface of the apical membrane. Third, the newly formed CO2 and H 2 O enter the proximal-tubule cell. Fourth, cytoplasmic CA II accelerates the regeneration of H + and HCO 3 - . Finally, this HCO 3 - exits the proximal-tubule cell across the basolateral membrane, completing the movement of HCO 3 - from lumen to blood. In the portion of the proximal tubule furthest downstream from the glomerulus, the S3 segment, the efflux of HCO 3 - across the basolateral membrane is mediated by both a Cl - /HCO 3 - exchanger and an electrogenic Na + :HCO 3 - cotransporter (Nakhoul et al. 1990). However, in more proximal segments of the proximal tubule (S2 and S1 segments), where the bulk of HCO3 - is in fact reabsorbed, the relative contribution of Cl - /HCO 3 - exchange decreases and that of electrogenic Na + :HCO 3 - cotransport increases (Kondo and Frömter, 1987). All told, the proximal tubule reabsorbs some 90 % of the filtered HCO3 - and 70 % of the filtered Na + . The electrogenic Na + :HCO 3 - cotransporter carries the vast majority of this HCO3 - and approximately 25 % of the Na + . 263 The Journal of Experimental Biology 200, 263–268 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JEB0677 The electrogenic Na + :HCO 3 - cotransporter (symporter) is the major transporter for HCO 3 - reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na + reabsorption. We expression-cloned the salamander renal electrogenic N a + :B icarbonate C otransporter (NBC) in Xenopus laevis oocytes. After injecting poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectrodes to monitor membrane potential (Vm ) and intracellular pH (pHi). All solutions contained ouabain to block the Na + /K + pump (P-ATPase). After applying 1.5 % CO 2 /10 mmol l -1 HCO 3 - (pH 7.5) and allowing pHi to stabilize from the CO 2 -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 HCO 3 - . 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 CO 2 /HCO 3 - 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 HCO 3 - 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. Key words: intracellular pH, acid–base balance, kidney, proximal tubule, bicarbonate transport. Summary Introduction THE RENAL ELECTROGENIC Na + :HCO 3 - COTRANSPORTER WALTER F. BORON 1, *, MATTHIAS A. HEDIGER 2 , EMILE L. BOULPAEP 1 AND MICHAEL F. ROMERO 1 1 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA and 2 Renal Division, Brigham and Women’s Hospital, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA *e-mail: Boronwf@maspo3.mas.yale.edu.