Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1996 Nov;71(5):2458–2466. doi: 10.1016/S0006-3495(96)79439-0

Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations.

J Zhao 1, B Zerhusen 1, J Xie 1, M L Drumm 1, P B Davis 1, J Ma 1
PMCID: PMC1233734  PMID: 8913585

Abstract

We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.

Full text

PDF
2458

Images in this article

2461FIGURE 3
on p.2461

Selected References

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

  1. Akabas M. H., Kaufmann C., Cook T. A., Archdeacon P. Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator. J Biol Chem. 1994 May 27;269(21):14865–14868. [PubMed] [Google Scholar]
  2. Anderson M. P., Gregory R. J., Thompson S., Souza D. W., Paul S., Mulligan R. C., Smith A. E., Welsh M. J. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science. 1991 Jul 12;253(5016):202–205. doi: 10.1126/science.1712984. [DOI] [PubMed] [Google Scholar]
  3. Bear C. E., Li C. H., Kartner N., Bridges R. J., Jensen T. J., Ramjeesingh M., Riordan J. R. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell. 1992 Feb 21;68(4):809–818. doi: 10.1016/0092-8674(92)90155-6. [DOI] [PubMed] [Google Scholar]
  4. Carson M. R., Welsh M. J. Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins. Biophys J. 1995 Dec;69(6):2443–2448. doi: 10.1016/S0006-3495(95)80113-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drumm M. L., Pope H. A., Cliff W. H., Rommens J. M., Marvin S. A., Tsui L. C., Collins F. S., Frizzell R. A., Wilson J. M. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell. 1990 Sep 21;62(6):1227–1233. doi: 10.1016/0092-8674(90)90398-x. [DOI] [PubMed] [Google Scholar]
  6. Egan M., Flotte T., Afione S., Solow R., Zeitlin P. L., Carter B. J., Guggino W. B. Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR. Nature. 1992 Aug 13;358(6387):581–584. doi: 10.1038/358581a0. [DOI] [PubMed] [Google Scholar]
  7. Ficker E., Taglialatela M., Wible B. A., Henley C. M., Brown A. M. Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science. 1994 Nov 11;266(5187):1068–1072. doi: 10.1126/science.7973666. [DOI] [PubMed] [Google Scholar]
  8. Frizzell R. A. Functions of the cystic fibrosis transmembrane conductance regulator protein. Am J Respir Crit Care Med. 1995 Mar;151(3 Pt 2):S54–S58. doi: 10.1164/ajrccm/151.3_Pt_2.S54. [DOI] [PubMed] [Google Scholar]
  9. Gadsby D. C., Nagel G., Hwang T. C. The CFTR chloride channel of mammalian heart. Annu Rev Physiol. 1995;57:387–416. doi: 10.1146/annurev.ph.57.030195.002131. [DOI] [PubMed] [Google Scholar]
  10. Gunderson K. L., Kopito R. R. Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating. J Biol Chem. 1994 Jul 29;269(30):19349–19353. [PubMed] [Google Scholar]
  11. Hanrahan J. W., Tabcharani J. A. Inhibition of an outwardly rectifying anion channel by HEPES and related buffers. J Membr Biol. 1990 Jun;116(1):65–77. doi: 10.1007/BF01871673. [DOI] [PubMed] [Google Scholar]
  12. Lopatin A. N., Makhina E. N., Nichols C. G. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature. 1994 Nov 24;372(6504):366–369. doi: 10.1038/372366a0. [DOI] [PubMed] [Google Scholar]
  13. Ma J., Mundiña-Weilenmann C., Hosey M. M., Ríos E. Dihydropyridine-sensitive skeletal muscle Ca channels in polarized planar bilayers. 1. Kinetics and voltage dependence of gating. Biophys J. 1991 Oct;60(4):890–901. doi: 10.1016/S0006-3495(91)82123-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ma J., Tasch J. E., Tao T., Zhao J., Xie J., Drumm M. L., Davis P. B. Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein. J Biol Chem. 1996 Mar 29;271(13):7351–7356. doi: 10.1074/jbc.271.13.7351. [DOI] [PubMed] [Google Scholar]
  15. McDonough S., Davidson N., Lester H. A., McCarty N. A. Novel pore-lining residues in CFTR that govern permeation and open-channel block. Neuron. 1994 Sep;13(3):623–634. doi: 10.1016/0896-6273(94)90030-2. [DOI] [PubMed] [Google Scholar]
  16. Quinton P. M. Cystic fibrosis: a disease in electrolyte transport. FASEB J. 1990 Jul;4(10):2709–2717. doi: 10.1096/fasebj.4.10.2197151. [DOI] [PubMed] [Google Scholar]
  17. Reisin I. L., Prat A. G., Abraham E. H., Amara J. F., Gregory R. J., Ausiello D. A., Cantiello H. F. The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J Biol Chem. 1994 Aug 12;269(32):20584–20591. [PubMed] [Google Scholar]
  18. Riordan J. R., Rommens J. M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J. L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066–1073. doi: 10.1126/science.2475911. [DOI] [PubMed] [Google Scholar]
  19. Schwiebert E. M., Egan M. E., Hwang T. H., Fulmer S. B., Allen S. S., Cutting G. R., Guggino W. B. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell. 1995 Jun 30;81(7):1063–1073. doi: 10.1016/s0092-8674(05)80011-x. [DOI] [PubMed] [Google Scholar]
  20. Tabcharani J. A., Chang X. B., Riordan J. R., Hanrahan J. W. Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. Nature. 1991 Aug 15;352(6336):628–631. doi: 10.1038/352628a0. [DOI] [PubMed] [Google Scholar]
  21. Tabcharani J. A., Rommens J. M., Hou Y. X., Chang X. B., Tsui L. C., Riordan J. R., Hanrahan J. W. Multi-ion pore behaviour in the CFTR chloride channel. Nature. 1993 Nov 4;366(6450):79–82. doi: 10.1038/366079a0. [DOI] [PubMed] [Google Scholar]
  22. Takeshima H., Nishimura S., Matsumoto T., Ishida H., Kangawa K., Minamino N., Matsuo H., Ueda M., Hanaoka M., Hirose T. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature. 1989 Jun 8;339(6224):439–445. doi: 10.1038/339439a0. [DOI] [PubMed] [Google Scholar]
  23. Tao T., Xie J., Drumm M. L., Zhao J., Davis P. B., Ma J. Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel. Biophys J. 1996 Feb;70(2):743–753. doi: 10.1016/S0006-3495(96)79614-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tilly B. C., Winter M. C., Ostedgaard L. S., O'Riordan C., Smith A. E., Welsh M. J. Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers. J Biol Chem. 1992 May 15;267(14):9470–9473. [PubMed] [Google Scholar]
  25. Tsui L. C. The cystic fibrosis transmembrane conductance regulator gene. Am J Respir Crit Care Med. 1995 Mar;151(3 Pt 2):S47–S53. doi: 10.1164/ajrccm/151.3_Pt_2.S47. [DOI] [PubMed] [Google Scholar]
  26. Tu Q., Velez P., Cortes-Gutierrez M., Fill M. Surface charge potentiates conduction through the cardiac ryanodine receptor channel. J Gen Physiol. 1994 May;103(5):853–867. doi: 10.1085/jgp.103.5.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Welsh M. J., Smith A. E. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell. 1993 Jul 2;73(7):1251–1254. doi: 10.1016/0092-8674(93)90353-r. [DOI] [PubMed] [Google Scholar]
  28. Xie J., Drumm M. L., Ma J., Davis P. B. Intracellular loop between transmembrane segments IV and V of cystic fibrosis transmembrane conductance regulator is involved in regulation of chloride channel conductance state. J Biol Chem. 1995 Nov 24;270(47):28084–28091. doi: 10.1074/jbc.270.47.28084. [DOI] [PubMed] [Google Scholar]

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

RESOURCES