An electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain.
We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3' end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (alpharb1NBC) and rb2NBC (alpharb2NBC). alpharb1NBC labels an approximately 130-kDa protein predominantly from kidney, and alpharb2NBC labels an approximately 130-kDa protein predominantly from brain. alpharb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpharb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO(2)/10 mM HCO(-)(3) to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO(2)-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na(+) reverses the pH(i) increase and elicits a rapid depolarization. In the presence of 450 microM DIDS, removing external Na(+) has no effect on pH(i) and elicits a small hyperpolarization. The rate of the pH(i) decrease elicited by removing Na(+) is insensitive to removing external Cl(-). Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.
Bevensee
M O
MO
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA.
Schmitt
B M
BM
Choi
I
I
Romero
M F
MF
Boron
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D005260:Female; D015723:Gene Library; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D027982:Sodium-Bicarbonate Symporters; D014982:Xenopus laevis
D000906:Antibodies; D002352:Carrier Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D027982:Sodium-Bicarbonate Symporters