Two variants of the rat brain sodium-driven chloride bicarbonate exchanger (NCBE): developmental expression and addition of a PDZ motif.
Regulation of pH in the central nervous system is critical to normal brain function and response to pathophysiological conditions. Here we identify two novel variants of the sodium-driven chloride bicarbonate exchanger (NCBE) from brain. The developmental expression pattern seen by in situ hybridization for the 90-bp containing insert (insert A) reveals strong expression in spinal cord and brain beginning in embryonic development. High-level expression is seen in cerebellar Purkinje cells and principal cells in hippocampus. The variant missing a 39-bp insert at the 3' end (insert B) encodes a protein in which the deduced carboxyterminal three amino acids are replaced with a unique 21 amino acid stretch terminating in a PDZ motif. rb2NCBE, the PDZ motif-encoding variant, is more highly expressed in astrocytes than is rb1NCBE. Both variants are expressed at similar levels in neurons. Expression varies with age and cell type. The FLAG epitope was fused in-frame at the amino terminus and each variant was expressed using a retroviral vector to study subcellular localization. Both variants were associated with the plasma membrane, but rb2NCBE colocalized with actin filaments to a greater extent, suggesting the PDZ form may interact with the cytoskeleton, whereas rb1NCBE was more often seen in intracellular vesicles. The PDZ motif-containing variant was much more active in pH regulation, with the expected ionic dependence on Na+, HCO3- and Cl- when expressed in 3T3 cells. These results are a first step towards understanding the regulation of expression and activity of this transporter in the brain.
Giffard
Rona G
RG
Department of Anesthesia, S272, Stanford University, Stanford, CA 94305-5117, USA. rona.giffard@stanford.edu
Lee
Yong-Sun
YS
Ouyang
Yi-Bing
YB
Murphy
Sherry L
SL
Monyer
Hannah
H
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics; D004594:Electrophysiology / Q000379:methods; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization / Q000379:methods; D008168:Lung / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012964:Sodium / Q000494:pharmacology; D013154:Spleen / Q000378:metabolism; D014162:Transfection
D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D004251:DNA Transposable Elements; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C033652:postsynaptic density proteins; D000643:Ammonium Chloride; D012964:Sodium