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. 1999 Jun 15;340(Pt 3):737–743.

Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney.

Y Maulet 1, R C Lambert 1, S Mykita 1, J Mouton 1, M Partisani 1, Y Bailly 1, G Bombarde 1, A Feltz 1
PMCID: PMC1220306  PMID: 10359659

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi S., Uchida S., Ito H., Hata M., Hiroe M., Marumo F., Sasaki S. Two isoforms of a chloride channel predominantly expressed in thick ascending limb of Henle's loop and collecting ducts of rat kidney. J Biol Chem. 1994 Jul 1;269(26):17677–17683. [PubMed] [Google Scholar]
  2. Beyenbach K. W., Dantzler W. H. Comparative kidney tubule sources, isolation, perfusion, and function. Methods Enzymol. 1990;191:167–226. doi: 10.1016/0076-6879(90)91014-w. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  5. Deeds D. G., Sullivan L. P., Fenton R. A., Tucker J. M., Cuppage F. E. Function and structure of perfused bullfrog kidney. Am J Physiol. 1977 Nov;233(5):F481–F490. doi: 10.1152/ajprenal.1977.233.5.F481. [DOI] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fahlke C., Knittle T., Gurnett C. A., Campbell K. P., George A. L., Jr Subunit stoichiometry of human muscle chloride channels. J Gen Physiol. 1997 Jan;109(1):93–104. doi: 10.1085/jgp.109.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fujita N., Mori H., Yura T., Ishihama A. Systematic sequencing of the Escherichia coli genome: analysis of the 2.4-4.1 min (110,917-193,643 bp) region. Nucleic Acids Res. 1994 May 11;22(9):1637–1639. doi: 10.1093/nar/22.9.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gnionsahe A., Claire M., Koechlin N., Bonvalet J. P., Farman N. Aldosterone binding sites along nephron of Xenopus and rabbit. Am J Physiol. 1989 Jul;257(1 Pt 2):R87–R95. doi: 10.1152/ajpregu.1989.257.1.R87. [DOI] [PubMed] [Google Scholar]
  10. Hechenberger M., Schwappach B., Fischer W. N., Frommer W. B., Jentsch T. J., Steinmeyer K. A family of putative chloride channels from Arabidopsis and functional complementation of a yeast strain with a CLC gene disruption. J Biol Chem. 1996 Dec 27;271(52):33632–33638. doi: 10.1074/jbc.271.52.33632. [DOI] [PubMed] [Google Scholar]
  11. Huang M. E., Chuat J. C., Galibert F. A voltage-gated chloride channel in the yeast Saccharomyces cerevisiae. J Mol Biol. 1994 Sep 30;242(4):595–598. doi: 10.1006/jmbi.1994.1607. [DOI] [PubMed] [Google Scholar]
  12. Jentsch T. J., Günther W., Pusch M., Schwappach B. Properties of voltage-gated chloride channels of the ClC gene family. J Physiol. 1995 Jan;482:19S–25S. doi: 10.1113/jphysiol.1995.sp020560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jørgensen P. L. Purification of Na+,K+-ATPase: enzyme sources, preparative problems, and preparation from mammalian kidney. Methods Enzymol. 1988;156:29–43. doi: 10.1016/0076-6879(88)56005-6. [DOI] [PubMed] [Google Scholar]
  14. Kieferle S., Fong P., Bens M., Vandewalle A., Jentsch T. J. Two highly homologous members of the ClC chloride channel family in both rat and human kidney. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6943–6947. doi: 10.1073/pnas.91.15.6943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lambert R. C., Maulet Y., Dupont J. L., Mykita S., Craig P., Volsen S., Feltz A. Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture: probing Ca2+ channel structure and function with antisense oligonucleotides. Mol Cell Neurosci. 1996 Mar;7(3):239–246. doi: 10.1006/mcne.1996.0018. [DOI] [PubMed] [Google Scholar]
  17. Lambert R. C., Maulet Y., Mouton J., Beattie R., Volsen S., De Waard M., Feltz A. T-type Ca2+ current properties are not modified by Ca2+ channel beta subunit depletion in nodosus ganglion neurons. J Neurosci. 1997 Sep 1;17(17):6621–6628. doi: 10.1523/JNEUROSCI.17-17-06621.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lindenthal S., Schmieder S., Ehrenfeld J., Wills N. K. Cloning and functional expression of a ClC Cl- channel from the renal cell line A6. Am J Physiol. 1997 Oct;273(4 Pt 1):C1176–C1185. doi: 10.1152/ajpcell.1997.273.4.C1176. [DOI] [PubMed] [Google Scholar]
  19. Lorenz C., Pusch M., Jentsch T. J. Heteromultimeric CLC chloride channels with novel properties. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13362–13366. doi: 10.1073/pnas.93.23.13362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McPherson P. S., Campbell K. P. Solubilization and biochemical characterization of the high affinity [3H]ryanodine receptor from rabbit brain membranes. J Biol Chem. 1990 Oct 25;265(30):18454–18460. [PubMed] [Google Scholar]
  21. Middleton R. E., Pheasant D. J., Miller C. Purification, reconstitution, and subunit composition of a voltage-gated chloride channel from Torpedo electroplax. Biochemistry. 1994 Nov 15;33(45):13189–13198. doi: 10.1021/bi00249a005. [DOI] [PubMed] [Google Scholar]
  22. Miller C., White M. M. Dimeric structure of single chloride channels from Torpedo electroplax. Proc Natl Acad Sci U S A. 1984 May;81(9):2772–2775. doi: 10.1073/pnas.81.9.2772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Noguchi S., Mishina M., Kawamura M., Numa S. Expression of functional (Na+ + K+)-ATPase from cloned cDNAs. FEBS Lett. 1987 Dec 10;225(1-2):27–32. doi: 10.1016/0014-5793(87)81125-0. [DOI] [PubMed] [Google Scholar]
  24. Oberleithner H., Gassner B., Dietl P., Wang W. Amphibian nephron: isolated kidney and cell fusion. Methods Enzymol. 1990;192:710–733. doi: 10.1016/0076-6879(90)92104-l. [DOI] [PubMed] [Google Scholar]
  25. Reeves W. B., Andreoli T. E. Renal epithelial chloride channels. Annu Rev Physiol. 1992;54:29–50. doi: 10.1146/annurev.ph.54.030192.000333. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schmidt-Rose T., Jentsch T. J. Transmembrane topology of a CLC chloride channel. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7633–7638. doi: 10.1073/pnas.94.14.7633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sontheimer H., Becker C. M., Pritchett D. B., Schofield P. R., Grenningloh G., Kettenmann H., Betz H., Seeburg P. H. Functional chloride channels by mammalian cell expression of rat glycine receptor subunit. Neuron. 1989 May;2(5):1491–1497. doi: 10.1016/0896-6273(89)90195-5. [DOI] [PubMed] [Google Scholar]
  29. Steinmeyer K., Schwappach B., Bens M., Vandewalle A., Jentsch T. J. Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease. J Biol Chem. 1995 Dec 29;270(52):31172–31177. doi: 10.1074/jbc.270.52.31172. [DOI] [PubMed] [Google Scholar]
  30. Stoner L. C. Isolated perfused amphibian renal tubules: the diluting segment. Am J Physiol. 1977 Nov;233(5):F438–F444. doi: 10.1152/ajprenal.1977.233.5.F438. [DOI] [PubMed] [Google Scholar]
  31. Uchida S., Sasaki S., Furukawa T., Hiraoka M., Imai T., Hirata Y., Marumo F. Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla. J Biol Chem. 1993 Feb 25;268(6):3821–3824. [PubMed] [Google Scholar]
  32. Vandewalle A., Cluzeaud F., Bens M., Kieferle S., Steinmeyer K., Jentsch T. J. Localization and induction by dehydration of ClC-K chloride channels in the rat kidney. Am J Physiol. 1997 May;272(5 Pt 2):F678–F688. doi: 10.1152/ajprenal.1997.272.5.F678. [DOI] [PubMed] [Google Scholar]
  33. Zimniak L., Winters C. J., Reeves W. B., Andreoli T. E. Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels. Am J Physiol. 1996 Jun;270(6 Pt 2):F1066–F1072. doi: 10.1152/ajprenal.1996.270.6.F1066. [DOI] [PubMed] [Google Scholar]
  34. Zimniak L., Winters C. J., Reeves W. B., Andreoli T. E. Cl- channels in basolateral renal medullary vesicles. X. Cloning of a Cl- channel from rabbit outer medulla. Kidney Int. 1995 Dec;48(6):1828–1836. doi: 10.1038/ki.1995.481. [DOI] [PubMed] [Google Scholar]

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