Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Apr;78(4):1881–1894. doi: 10.1016/S0006-3495(00)76737-3

Gating of amiloride-sensitive Na(+) channels: subunit-subunit interactions and inhibition by the cystic fibrosis transmembrane conductance regulator.

B K Berdiev 1, V G Shlyonsky 1, K H Karlson 1, B A Stanton 1, I I Ismailov 1
PMCID: PMC1300782  PMID: 10733968

Abstract

In search of the structural basis for gating of amiloride-sensitive Na(+) channels, kinetic properties of single homo and heterooligomeric ENaCs formed by the subunits with individual truncated cytoplasmic domains were studied in a cell-free planar lipid bilayer reconstitution system. Our results identify the N-terminus of the alpha-subunit as a major determinant of kinetic behavior of both homooligomeric and heterooligomeric ENaCs, although the carboxy-terminal domains of beta- and gamma-ENaC subunits play important role(s) in modulation of the kinetics of heterooligomeric channels. We also found that the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits amiloride-sensitive channels, at least in part, by modulating their gating. Comparison of these data suggests that the modulatory effects of the beta- and gamma-ENaC subunits, and of the CFTR, may involve the same, or closely related, mechanism(s); namely, "locking" the heterooligomeric channels in their closed state. These mechanisms, however, do not completely override the gating mechanism of the alpha-channel.

Full Text

The Full Text of this article is available as a PDF (304.9 KB).

Selected References

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

  1. Adams C. M., Snyder P. M., Welsh M. J. Interactions between subunits of the human epithelial sodium channel. J Biol Chem. 1997 Oct 24;272(43):27295–27300. doi: 10.1074/jbc.272.43.27295. [DOI] [PubMed] [Google Scholar]
  2. Awayda M. S., Ismailov I. I., Berdiev B. K., Benos D. J. A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers. Am J Physiol. 1995 Jun;268(6 Pt 1):C1450–C1459. doi: 10.1152/ajpcell.1995.268.6.C1450. [DOI] [PubMed] [Google Scholar]
  3. Awayda M. S., Tousson A., Benos D. J. Regulation of a cloned epithelial Na+ channel by its beta- and gamma-subunits. Am J Physiol. 1997 Dec;273(6 Pt 1):C1889–C1899. doi: 10.1152/ajpcell.1997.273.6.C1889. [DOI] [PubMed] [Google Scholar]
  4. Baker E., Jeunemaitre X., Portal A. J., Grimbert P., Markandu N., Persu A., Corvol P., MacGregor G. Abnormalities of nasal potential difference measurement in Liddle's syndrome. J Clin Invest. 1998 Jul 1;102(1):10–14. doi: 10.1172/JCI1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berdiev B. K., Karlson K. H., Jovov B., Ripoll P. J., Morris R., Loffing-Cueni D., Halpin P., Stanton B. A., Kleyman T. R., Ismailov I. I. Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel. Biophys J. 1998 Nov;75(5):2292–2301. doi: 10.1016/S0006-3495(98)77673-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bixby K. A., Nanao M. H., Shen N. V., Kreusch A., Bellamy H., Pfaffinger P. J., Choe S. Zn2+-binding and molecular determinants of tetramerization in voltage-gated K+ channels. Nat Struct Biol. 1999 Jan;6(1):38–43. doi: 10.1038/4911. [DOI] [PubMed] [Google Scholar]
  7. Briel M., Greger R., Kunzelmann K. Cl- transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC. J Physiol. 1998 May 1;508(Pt 3):825–836. doi: 10.1111/j.1469-7793.1998.825bp.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Canessa C. M., Horisberger J. D., Rossier B. C. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 1993 Feb 4;361(6411):467–470. doi: 10.1038/361467a0. [DOI] [PubMed] [Google Scholar]
  9. Canessa C. M., Schild L., Buell G., Thorens B., Gautschi I., Horisberger J. D., Rossier B. C. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature. 1994 Feb 3;367(6462):463–467. doi: 10.1038/367463a0. [DOI] [PubMed] [Google Scholar]
  10. Chabot H., Vives M. F., Dagenais A., Grygorczyk C., Berthiaume Y., Grygorczyk R. Downregulation of epithelial sodium channel (ENaC) by CFTR co-expressed in Xenopus oocytes is independent of Cl- conductance. J Membr Biol. 1999 Jun 1;169(3):175–188. doi: 10.1007/s002329900529. [DOI] [PubMed] [Google Scholar]
  11. Chang S. S., Grunder S., Hanukoglu A., Rösler A., Mathew P. M., Hanukoglu I., Schild L., Lu Y., Shimkets R. A., Nelson-Williams C. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet. 1996 Mar;12(3):248–253. doi: 10.1038/ng0396-248. [DOI] [PubMed] [Google Scholar]
  12. Cheng C., Prince L. S., Snyder P. M., Welsh M. J. Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis. J Biol Chem. 1998 Aug 28;273(35):22693–22700. doi: 10.1074/jbc.273.35.22693. [DOI] [PubMed] [Google Scholar]
  13. Coscoy S., Lingueglia E., Lazdunski M., Barbry P. The Phe-Met-Arg-Phe-amide-activated sodium channel is a tetramer. J Biol Chem. 1998 Apr 3;273(14):8317–8322. doi: 10.1074/jbc.273.14.8317. [DOI] [PubMed] [Google Scholar]
  14. Firsov D., Gautschi I., Merillat A. M., Rossier B. C., Schild L. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J. 1998 Jan 15;17(2):344–352. doi: 10.1093/emboj/17.2.344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Firsov D., Schild L., Gautschi I., Mérillat A. M., Schneeberger E., Rossier B. C. Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15370–15375. doi: 10.1073/pnas.93.26.15370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gründer S., Firsov D., Chang S. S., Jaeger N. F., Gautschi I., Schild L., Lifton R. P., Rossier B. C. A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel. EMBO J. 1997 Mar 3;16(5):899–907. doi: 10.1093/emboj/16.5.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hansson J. H., Nelson-Williams C., Suzuki H., Schild L., Shimkets R., Lu Y., Canessa C., Iwasaki T., Rossier B., Lifton R. P. Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet. 1995 Sep;11(1):76–82. doi: 10.1038/ng0995-76. [DOI] [PubMed] [Google Scholar]
  18. Hansson J. H., Schild L., Lu Y., Wilson T. A., Gautschi I., Shimkets R., Nelson-Williams C., Rossier B. C., Lifton R. P. A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11495–11499. doi: 10.1073/pnas.92.25.11495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hopf A., Schreiber R., Mall M., Greger R., Kunzelmann K. Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations. J Biol Chem. 1999 May 14;274(20):13894–13899. doi: 10.1074/jbc.274.20.13894. [DOI] [PubMed] [Google Scholar]
  20. Hoshi T., Zagotta W. N., Aldrich R. W. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990 Oct 26;250(4980):533–538. doi: 10.1126/science.2122519. [DOI] [PubMed] [Google Scholar]
  21. Ismailov I. I., Awayda M. S., Berdiev B. K., Bubien J. K., Lucas J. E., Fuller C. M., Benos D. J. Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. J Biol Chem. 1996 Jan 12;271(2):807–816. doi: 10.1074/jbc.271.2.807. [DOI] [PubMed] [Google Scholar]
  22. Ismailov I. I., Awayda M. S., Jovov B., Berdiev B. K., Fuller C. M., Dedman J. R., Kaetzel M., Benos D. J. Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1996 Mar 1;271(9):4725–4732. doi: 10.1074/jbc.271.9.4725. [DOI] [PubMed] [Google Scholar]
  23. Ismailov I. I., Berdiev B. K., Shlyonsky V. G., Fuller C. M., Prat A. G., Jovov B., Cantiello H. F., Ausiello D. A., Benos D. J. Role of actin in regulation of epithelial sodium channels by CFTR. Am J Physiol. 1997 Apr;272(4 Pt 1):C1077–C1086. doi: 10.1152/ajpcell.1997.272.4.C1077. [DOI] [PubMed] [Google Scholar]
  24. Ismailov I. I., Shlyonsky V. G., Alvarez O., Benos D. J. Cation permeability of a cloned rat epithelial amiloride-sensitive Na+ channel. J Physiol. 1997 Oct 15;504(Pt 2):287–300. doi: 10.1111/j.1469-7793.1997.287be.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ismailov I. I., Shlyonsky V. G., Serpersu E. H., Fuller C. M., Cheung H. C., Muccio D., Berdiev B. K., Benos D. J. Peptide inhibition of ENaC. Biochemistry. 1999 Jan 5;38(1):354–363. doi: 10.1021/bi981979s. [DOI] [PubMed] [Google Scholar]
  26. Jovov B., Ismailov I. I., Berdiev B. K., Fuller C. M., Sorscher E. J., Dedman J. R., Kaetzel M. A., Benos D. J. Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels. J Biol Chem. 1995 Dec 8;270(49):29194–29200. doi: 10.1074/jbc.270.49.29194. [DOI] [PubMed] [Google Scholar]
  27. Kizer N., Guo X. L., Hruska K. Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):1013–1018. doi: 10.1073/pnas.94.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kosari F., Sheng S., Li J., Mak D. O., Foskett J. K., Kleyman T. R. Subunit stoichiometry of the epithelial sodium channel. J Biol Chem. 1998 May 29;273(22):13469–13474. doi: 10.1074/jbc.273.22.13469. [DOI] [PubMed] [Google Scholar]
  29. Kunzelmann K., Kiser G. L., Schreiber R., Riordan J. R. Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator. FEBS Lett. 1997 Jan 6;400(3):341–344. doi: 10.1016/s0014-5793(96)01414-7. [DOI] [PubMed] [Google Scholar]
  30. Li M., Jan Y. N., Jan L. Y. Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. Science. 1992 Aug 28;257(5074):1225–1230. doi: 10.1126/science.1519059. [DOI] [PubMed] [Google Scholar]
  31. Ling B. N., Zuckerman J. B., Lin C., Harte B. J., McNulty K. A., Smith P. R., Gomez L. M., Worrell R. T., Eaton D. C., Kleyman T. R. Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line. J Biol Chem. 1997 Jan 3;272(1):594–600. doi: 10.1074/jbc.272.1.594. [DOI] [PubMed] [Google Scholar]
  32. Lingueglia E., Voilley N., Waldmann R., Lazdunski M., Barbry P. Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett. 1993 Feb 22;318(1):95–99. doi: 10.1016/0014-5793(93)81336-x. [DOI] [PubMed] [Google Scholar]
  33. Mall M., Hipper A., Greger R., Kunzelmann K. Wild type but not deltaF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes. FEBS Lett. 1996 Feb 26;381(1-2):47–52. doi: 10.1016/0014-5793(96)00079-8. [DOI] [PubMed] [Google Scholar]
  34. Prince L. S., Welsh M. J. Cell surface expression and biosynthesis of epithelial Na+ channels. Biochem J. 1998 Dec 15;336(Pt 3):705–710. doi: 10.1042/bj3360705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prince L. S., Welsh M. J. Effect of subunit composition and Liddle's syndrome mutations on biosynthesis of ENaC. Am J Physiol. 1999 Jun;276(6 Pt 1):C1346–C1351. doi: 10.1152/ajpcell.1999.276.6.C1346. [DOI] [PubMed] [Google Scholar]
  36. Rosenberg R. L., East J. E. Cell-free expression of functional Shaker potassium channels. Nature. 1992 Nov 12;360(6400):166–169. doi: 10.1038/360166a0. [DOI] [PubMed] [Google Scholar]
  37. Schild L., Canessa C. M., Shimkets R. A., Gautschi I., Lifton R. P., Rossier B. C. A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5699–5703. doi: 10.1073/pnas.92.12.5699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shen N. V., Chen X., Boyer M. M., Pfaffinger P. J. Deletion analysis of K+ channel assembly. Neuron. 1993 Jul;11(1):67–76. doi: 10.1016/0896-6273(93)90271-r. [DOI] [PubMed] [Google Scholar]
  39. Shen N. V., Pfaffinger P. J. Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K+ channel subunit proteins. Neuron. 1995 Mar;14(3):625–633. doi: 10.1016/0896-6273(95)90319-4. [DOI] [PubMed] [Google Scholar]
  40. Shimkets R. A., Warnock D. G., Bositis C. M., Nelson-Williams C., Hansson J. H., Schambelan M., Gill J. R., Jr, Ulick S., Milora R. V., Findling J. W. Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell. 1994 Nov 4;79(3):407–414. doi: 10.1016/0092-8674(94)90250-x. [DOI] [PubMed] [Google Scholar]
  41. Stutts M. J., Canessa C. M., Olsen J. C., Hamrick M., Cohn J. A., Rossier B. C., Boucher R. C. CFTR as a cAMP-dependent regulator of sodium channels. Science. 1995 Aug 11;269(5225):847–850. doi: 10.1126/science.7543698. [DOI] [PubMed] [Google Scholar]
  42. Stutts M. J., Rossier B. C., Boucher R. C. Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics. J Biol Chem. 1997 May 30;272(22):14037–14040. doi: 10.1074/jbc.272.22.14037. [DOI] [PubMed] [Google Scholar]
  43. Valentijn J. A., Fyfe G. K., Canessa C. M. Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes. J Biol Chem. 1998 Nov 13;273(46):30344–30351. doi: 10.1074/jbc.273.46.30344. [DOI] [PubMed] [Google Scholar]
  44. Verrall S., Hall Z. W. The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly. Cell. 1992 Jan 10;68(1):23–31. doi: 10.1016/0092-8674(92)90203-o. [DOI] [PubMed] [Google Scholar]
  45. Zagotta W. N., Hoshi T., Aldrich R. W. Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science. 1990 Oct 26;250(4980):568–571. doi: 10.1126/science.2122520. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES