Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells.
We previously reported a 120-kDa phosphoprotein that translocated from cytosol to the apical membrane of gastric parietal cells in association with stimulation of HCl secretion. To determine the molecular identity of the protein, we performed molecular cloning and expression of the protein. Immunoblot analysis showed that this protein was highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. We named this protein "parchorin" based on its highest enrichment in parietal cells and choroid plexus. We obtained cDNA for parchorin from rabbit choroid plexus coding a protein consisting of 637 amino acids with a predicted molecular mass of 65 kDa. The discrepancy in size on 6% SDS-polyacrylamide gel electrophoresis is considered to be due to its highly acidic nature (pI = 4.18), because COS-7 cells transfected with parchorin cDNA produced a protein with apparent molecular mass of 120 kDa on 6% SDS-polyacrylamide gel electrophoresis. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels (CLIC), especially the chloride channel from bovine kidney, p64, in the C-terminal 235 amino acids. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, existed mainly in the cytosol. Furthermore, when Cl(-) efflux from the cell was elicited, GFP-parchorin translocated to the plasma membrane. These results suggest that parchorin generally plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport.
Nishizawa
T
T
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
Nagao
T
T
Iwatsubo
T
T
Forte
J G
JG
Urushidani
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001834:Body Water / Q000378:metabolism*; D018118:Chloride Channels / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D002831:Choroid Plexus / Q000737:chemistry*; D003001:Cloning, Molecular; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000737:chemistry*; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification*; D011817:Rabbits
D018118:Chloride Channels; D002712:Chlorides; D010750:Phosphoproteins; C407265:parchorin protein, Oryctolagus cuniculus