THE ELECTROGENIC NA HCO3 COTRANSPORTER

The electrogenic Na/HCO3 cotransporter (symporter) is the major HCO3- transporter of the renal proximal tubule (PT), located at the basolate ral membrane (BLM), and also plays a noteworthy role in Na+ reabsorpti on, HCO3 transporters are important for regulation of intracellular pH (pH(i)) in most cells and also thereby regulate blood pH. This electr ogenic Na/HCO3 cotransporter was first discovered using perfused Ambys toma tigrinum (salamander) renal, proximal tubules. This novel cotrans porter mediates the movement of one Na+ ion with several HCO3- ions, m aking it electrogenic, is blocked by stilbene compounds, but does not depend on intra- or extracellular Cl-. This and similar cotransporters have been found in a number of tissues and cell types. Recently, we u sed Xenopus laevis oocytes to expression clone the salamander renal el ectrogenic Na Bicarbonate Cotransporter (NBC). Using microelectrodes t o monitor membrane potential (V-m) and intracellular pH (pH(i)), we fo llowed oocyte expresion after injecting poly (A)+, fractionated poly ( A)(+), or cRNA. All experimental solutions contained 100 mu M ouabain to block the Na+/K+ pump. Our expression assay was to apply 1.5% CO2/1 0 mM HCO3- (pH 7.5), allow pH(i) to stabilize from the CO2-induced aci dification, and then remove bath Na+. Removing bath Na+ from native oo cytes and water-injected controls, hyperpolarized the oocytes by simil ar to 5 mV and had no effect on pH(i). However, for oocytes injected w ith poly (A)(+) RNA, removing Na+ transiently depolarized the cell by similar to 10 mV and caused pH(i) to decrease; both effects were block ed by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO. Electrophoretic fractionation of the poly (A)(+) RNA, enriched t he expression signal. From the optimal expression-fraction, we constru cted a size-selected cDNA library in pSPORT1. Screening our Ambystoma library yielded a single clone (aNBC). We could detect expression 3 da ys after injection of NBC cRNA. In aNBC-expresssing oocytes, adding CO 2/HCO(3)(-)elicited a large (> 50mV) and rapid hyperpolarization, foll owed by a partial relaxation as pH(i) stabilized. Na+ removal in CO2/H CO3similar to depolarized the cell by >40mV and decreased pH(i), aNBC encodes a protein of 1035 amino acids with several putative membrane-s panning domains, and has a low level of amino-acid homology (similar t o 30% to the AE family of Cl-HCO3 exchangers. aNBC is the first member of a new family of Na+-linked HCO3- transporters and, together with t he AE family, defines a new superfamily of HCO3- transporters. Using a NBC to screen a rat-kidney cDNA library, we identified a full-length c DNA clone (rNBC), rNBC encodes a protein of 1035 amino acids, is 86% i dentical to aNBC, and can be functionally expressed in oocytes.