Cloning and functional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney.
We have recently cloned the renal electrogenic Na(+)-bicarbonate contransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na(+)-bicarbonate cotransporter. NBC constitutes the major route for HCO3- reabsorption and assists in Na+ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in lambda gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of approximately 500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (approximately 30%) to the AE family of Cl-/HCO3- exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na(+)-HCO3- contransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO2/10 mM HCO3- elicits a hyperpolarization of > 50 mV and a rapid decrease of intracellular pH (pHi), followed by an increase in pHi. Subsequent removal of Na+ in the presence of CO2/HCO3- causes a depolarization of > 50 mV and a concomitant decrease of pHi. Thus rNBC is in the same newly identified family of Na(+)-linked HCO3- transporters as is aNBC.
Romero
M F
MF
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Fong
P
P
Berger
U V
UV
Hediger
M A
MA
Boron
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D007668:Kidney / Q000737:chemistry*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D027982:Sodium-Bicarbonate Symporters; D014162:Transfection; D014982:Xenopus laevis
D002352:Carrier Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D027982:Sodium-Bicarbonate Symporters