Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1.
CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate Cl(-) approximately Br(-) > I(-) > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK(a) of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.
Dowland
L K
LK
Renal Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Luyckx
V A
VA
Enck
A H
AH
Leclercq
B
B
Yu
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D014981:Xenopus
C097503:CLC-5 chloride channel; D018118:Chloride Channels; D018076:DNA, Complementary