THE RENAL ELECTROGENIC NA+HCO3- COTRANSPORTER

The electrogenic Na+:HCO3- cotransporter (symporter) is the major tran sporter for HCO3- reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na+ reabso rption. We expression-cloned the salamander renal electrogenic Na+:Bic arbonate Cotransporter (NBC) in Xenopus laevis oocytes, After injectin g poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectr odes to monitor membrane potential (V-m) and intracellular pH (pHi). A ll solutions contained ouabain to block the Na+/K+ pump (P-ATPase), Af ter applying 1.5 % CO2/10 mmol l(-1) HCO3- (pH 7.5) and allowing pHi t o stabilize from the CO2-induced acidification, we removed Na+. In nat ive oocytes or water-injected controls, removing Na+ hyperpolarized th e cell by -5 mV and had no effect on pHi. In oocytes injected with pol y(A)+ RNA, removing Na+ transiently depolarized the cell by -10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocy ano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. We enriched th e signal by electrophoretic fractionation of the poly(A)+ RNA, and con structed a size-selected cDNA library in pSPORT1 using the optimal fra ction. Screening the Ambystoma library yielded a single clone (aNBC). Expression was first obvious 3 days after injection of NBC cRNA, Addin g CO2/HCO3- induced a large (>50 mV) and rapid hyperpolarization, foll owed by a partial relaxation as pill stabilized. Subsequent Na+ remova l 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 d omains, aNBC is the first member of a new family of Na+-linked HCO3- t ransporters, We used aNBC to screen a rat kidney cDNA library, and ide ntified a full-length cDNA clone (rNBC) that encodes a protein of 1035 amino acids, rNBC is 86% identical to aNBC and can be functionally ex pressed in oocytes.