The renal electrogenic Na+:HCO-3 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 (Vm) 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.
Boron
V F
VF
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA. Boronwf@maspo3.mas.yale.edu
Hediger
M A
MA
Boulpaep
E L
EL
Romero
M F
MF
D000557:Ambystoma / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008564:Membrane Potentials; D008845:Microinjections; D009865:Oocytes; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D001639:Bicarbonates; D002352:Carrier Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D027982:Sodium-Bicarbonate Symporters; D012964:Sodium