Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney.
ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K. The cDNA encodes a 77 kDa protein presenting 62% similarity with human ClC-Kb. The protein is monoglycosylated and is expressed primarily in the Xenopus kidney. It is localized in the basolateral membranes of proximal convoluted tubules of the nephron and in the apical region of the diluting segments. Heterologous expression of xClC-K in HEK-293 cells showed that the full-length protein is glycosylated and targeted to the cell membrane, but no associated Cl- current could be observed with the patch-clamp recording technique. N-glycosylation of both the native kidney channel and the recombinant protein expressed in HEK-293 conferred on them anomalous behaviour in denaturing PAGE, which is indicative of strong interactions at the extracellular side of the plasma membrane. The expression of ClC-K channels in both mesonephric and metanephric kidneys will permit further comparative physiological studies of Cl- permeabilities at the molecular level.
Maulet
Y
Y
Laboratoire de Neurobiologie Cellulaire, UPR 9009 CNRS, 5 rue Blaise Pascal, F-67084 Strasbourg, France. maulet@neurochem.u-strasbg.fr
Lambert
R C
RC
Mykita
S
S
Mouton
J
J
Partisani
M
M
Bailly
Y
Y
Bombarde
G
G
Feltz
A
A
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D005260:Female; D006031:Glycosylation; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009928:Organ Specificity; D018408:Patch-Clamp Techniques; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
C489932:CLCNKA protein, human; D018118:Chloride Channels; D002712:Chlorides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C489934:clcnka protein, Xenopus; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase