Molecular Cloning and Characterization of an Intracellular Chloride Channel in the Proximal Tubule Cell Line, LLC-PK1

Abstract

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 ؊ Ϸ 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.

Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates

“…The xtTRPV1 gene was cloned into pENTR/D-Topo (Invitrogen) and then subcloned into the pVenus-NLS vector for transfection into HeLa cells or into the pOX(ϩ) (15) vector for oocyte expression. pVenus-NLS was constructed by introducing five mutations (F46L/ F64L/M153T/V163A/S175G) into pYFP-C1 (Clontech) as previously described (16) and by inserting the oligonucleotide (TCCGGAGATCCAAAGAAAAAGAGAAAAGTAGATCC-AAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGA-GAAAGGTAAGATCT) coding three copies of the nuclear localization signal of the simian virus 40 large T-antigen (17) between the BspEI and BglII sites.…”

Expression of claudin-7 and -8 along the mouse nephron

“…Recombinant plasmid DNA, or vector alone, was transiently transfected into human embryonic kidney (HEK-293) cells, which were then harvested, and 100,000-g membranes were isolated. Reducing SDS-PAGE and immunoblotting were performed by standard methods, as described previously (9).…”

Claudin-8 Expression in Madin-Darby Canine Kidney Cells Augments the Paracellular Barrier to Cation Permeation

“…Immunohistochemistry-To detect tight junction protein expression by immunoblotting, cultured cells were first homogenized and fractionated by centrifugation at 100,000 ϫ g as described previously (24). No claudin-8 was ever detectable in the soluble fraction (100,000 ϫ g supernatant).…”

“…1C) with a rabbit anti-claudin-8 antibody (933) at 1:100 dilution, all in the presence of 0.3% Triton X-100 using protocols described previously (24). Affinitypurified rabbit polyclonal antibody 933 was raised against the claudin-8 C-terminal peptide, CQRSFHAEKRSPSIYSKSQYV (25).…”

“…Lines-A 675-bp DNA fragment containing the mouse claudin-8 coding sequence except for the initiation codon was amplified by PCR from a cDNA clone (IMAGE ID: 162549) and inserted into a FLAG epitope tag shuttle vector based on pcDNA3 (24) so that the claudin-8 N terminus was fused in-frame with a sequence encoding MDYKDDDDKGS (the FLAG octapeptide tag is underlined followed by a glycine-serine linker encoding BamHI restriction site). The entire coding region was then excised and cloned into the retroviral Tet response vector, pRevTRE (Clontech, Palo Alto, CA) to obtain the plasmid, pRev-mCLDN8-NFL.…”