Molecular Cloning and Characterization of the Cl− Pump-Associated 55-kDa Protein in Rat Brain

• Published in: Biochemical and biophysical research communications 2001-11, Vol.289 (2), p.363-371
• Main Authors: Kitagawa, Kaori, Yagyu, Kyoko, Yamamoto, Akitsugu, Hattori, Naoki, Omori, Kyoko, Zeng, Xun-Ting, Inagaki, Chiyoko
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Amino Acid Sequence; Animals; Anion Transport Proteins; antisense oligonucleotide; Base Sequence; Blotting, Northern; Blotting, Western; brain; Brain - enzymology; cDNA cloning; Cell Membrane - enzymology; Cell Membrane - metabolism; Cells, Cultured; Cerebellum - metabolism; Chlorine - metabolism; Cloning, Molecular; Cl− pump; Cl−-ATPase; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase - metabolism; hippocampal neuron; Hippocampus - embryology; Hippocampus - enzymology; Immunohistochemistry; Microscopy, Fluorescence; Molecular Sequence Data; Neurons - enzymology; Neurons - metabolism; Oligonucleotides, Antisense - chemistry; Open Reading Frames; Protein Processing, Post-Translational; Rats; Rats, Wistar; Recombinant Fusion Proteins - metabolism; Signal Transduction; Telencephalon - enzymology; Tissue Distribution; Transfection
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.5997

The Cl−-ATPase/pump in the plasma membrane of the rat brain is a candidate for active outwardly directed Cl− translocating systems. We recently isolated a Cl− pump, 520- or 580-kDa protein complex, which consisted of 51-, 55-, 60-, and 62-kDa proteins. In this study, we cloned a cDNA encoding a 55-kDa glycoprotein, designated as ClP55, which contained an open reading frame of 1512 base pairs encoding a protein of 504 amino acids including a signal peptide of 28 amino acids. Northern and Western blot analyses demonstrated expression of ClP55 mainly in the cerebrum. Application of antisense phosphorothioate oligonucleotides to cultured neurons resulted in a marked increase in the intracellular Cl− concentration ([Cl−]i). Immunohistochemical analysis indicated that ClP55 was localized to the plasma membranes of neurons such as hippocampal pyramidal neurons and cerebellar Purkinje cells. Taken together, these results suggest that ClP55 is one of the Cl− pump subunits responsible for Cl− pump activity.