Nucleotides bind to the C-terminus of ClC-5

Leigh Wellhauser

Hsin Kuo

Fiona Stratford

Mohabir Ramjeesingh

*Programme in Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8

†Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8

‡Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 1A8

^{To whom correspondence should be addressed, at Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8 (email}ac.sdikkcis@raeb).

Received 2006 Jan 23; Revised 2006 May 9; Accepted 2006 May 11.

Abstract

Mutations in ClC-5 (chloride channel 5), a member of the ClC family of chloride ion channels and antiporters, have been linked to Dent's disease, a renal disease associated with proteinuria. Several of the disease-causing mutations are premature stop mutations which lead to truncation of the C-terminus, pointing to the functional significance of this region. The C-terminus of ClC-5, like that of other eukaryotic ClC proteins, is cytoplasmic and contains a pair of CBS (cystathionine β-synthase) domains connected by an intervening sequence. The presence of CBS domains implies a regulatory role for nucleotide interaction based on studies of other unrelated proteins bearing these domains [Ignoul and Eggermont (2005) Am. J. Physiol. Cell Physiol. 289, C1369–C1378; Scott, Hawley, Green, Anis, Stewart, Scullion, Norman and Hardie (2004) J. Clin. Invest. 113, 274–284]. However, to date, there has been no direct biochemical or biophysical evidence to support nucleotide interaction with ClC-5. In the present study, we have expressed and purified milligram quantities of the isolated C-terminus of ClC-5 (CIC-5 Ct). CD studies show that the protein is compact, with predominantly α-helical structure. We determined, using radiolabelled ATP, that this nucleotide binds the folded protein with low affinity, in the millimolar range, and that this interaction can be competed with 1 μM AMP. CD studies show that binding of these nucleotides causes no significant change in secondary structure, consistent with a model wherein these nucleotides bind to a preformed site. However, both nucleotides induce an increase in thermal stability of ClC-5 Ct, supporting the suggestion that both nucleotides interact with and modify the biophysical properties of this protein.

Keywords: CD, chloride channel 5 (ClC-5), chloride ion channel, cystathionine β-synthase, nucleotide, protein purification

Abbreviations: CBS domain, cystathionine β-synthase domain, CFTR, cystic fibrosis transmembrane conductance regulator, ClC-5, chloride channel 5, ClC-5 Ct, C-terminus of ClC-5, hCLC-5, human ClC-5, IPTG, isopropyl β-D-thiogalactoside

Abstract

Acknowledgments

This research was supported primarily through a grant to C. E. B. from the Canadian Institutes of Health Research and in part through a grant to C. M. D. from the Canadian Cystic Fibrosis Foundation. L. W. and W. L. were supported by scholarships awarded by Natural Sciences and Engineering Research Council. H.-H. K. holds a Research Training Award from the Research Institute, Hospital for Sick Children (Toronto, ON, Canada). We also acknowledge contributions by Dr P. Lynne Howell (Head of the Programme in Structural Biology and Biochemistry, Hospital for Sick Children) in designing the solubility and stability screens for this protein, tools that were essential in optimizing the yield of protein for biophysical studies.

Acknowledgments

References

1. Thakker R. V. Chloride channels in renal disease. Adv. Nephrol. Necker Hosp. 1999;29:289–298.[PubMed]
2. George AL., Jr Chloride channels and endocytosis: ClC-5 makes a dent. Proc. Natl. Acad. Sci. U.S.A. 1998;95:7843–7845.[Google Scholar]
3. Jentsch T. J. Chloride transport in the kidney: lessons from human disease and knockout mice. J. Am. Soc. Nephrol. 2005;16:1549–1561.[PubMed]
4. Christensen E. I., Devuyst O., Dom G., Nielsen R., Van der Smissen P., Verroust P., Leruth M., Guggino W. B., Courtoy P. J. Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules. Proc. Natl. Acad. Sci. U.S.A. 2003;100:8472–8477.
5. Piwon N., Gunther W., Schwake M., Bosl M. R., Jentsch T. J. ClC-5 Cl^{-channel disruption impairs endocytosis in a mouse model for Dent's disease.}Nature (London) 2000;408:369–373.[PubMed]
6. Hara-Chikuma M., Wang Y., Guggino S. E., Guggino W. B., Verkman A. S. Impaired acidification in early endosomes of ClC-5 deficient proximal tubule. Biochem. Biophys. Res. Commun. 2005;329:941–946.[PubMed]
7. Gunther W., Piwon N., Jentsch T. J. The ClC-5 chloride channel knock-out mouse – an animal model for Dent's disease. Pflugers Arch. 2003;445:456–462.[PubMed]
8. Mohammad-Panah R., Harrison R., Dhani S., Ackerley C., Huan L. J., Wang Y., Bear C. E. The chloride channel ClC-4 contributes to endosomal acidification and trafficking. J. Biol. Chem. 2003;278:29267–29277.[PubMed]
9. van Weert A. W., Dunn K. W., Gueze H. J., Maxfield F. R., Stoorvogel W. Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump. J. Cell Biol. 1995;130:821–834.
10. Jentsch T. J., Neagoe I., Scheel O. CLC chloride channels and transporters. Curr. Opin. Neurobiol. 2005;15:319–325.[PubMed]
11. Dutzler R., Campbell E. B., Cadene M., Chait B. T., MacKinnon R. X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature (London) 2002;415:287–294.[PubMed]
12. Jordt S. E., Jentsch T. J. Molecular dissection of gating in the ClC-2 chloride channel. EMBO J. 1997;16:1582–1592.
13. Estevez R., Pusch M., Ferrer-Costa C., Orozco M., Jentsch T. J. Functional and structural conservation of CBS domains from CLC chloride channels. J. Physiol. 2004;557:363–378.
14. Hebeisen S., Fahlke CCarboxy-terminal truncations modify the outer pore vestibule of muscle chloride channels. Biophys. J. 2005;89:1710–1720.[Google Scholar]
15. Denton J., Nehrke K., Yin X., Beld A., Strange KAltered gating and regulation of a carboxy-terminus ClC channel mutant expressed in the C. elegans oocyte. Am. J. Physiol. Cell Physiol. 2005;290:C1109–C1118.[PubMed][Google Scholar]
16. Ignoul S., Eggermont JCBS domains: structure, function, and pathology in human proteins. Am. J. Physiol. Cell Physiol. 2005;289:C1369–C1378.[PubMed][Google Scholar]
17. Meyer S., Dutzler RCrystal structure of the cytoplasmic domain of the chloride channel ClC-0. Structure. 2006;14:299–307.[PubMed][Google Scholar]
18. Scott J. W., Hawley S. A., Green K. A., Anis M., Stewart G., Scullion G. A., Norman D. G., Hardie D. G. CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J. Clin. Invest. 2004;113:274–284.
19. Bennetts B., Rychkov G. Y., Ng H. L., Morton C. J., Stapleton D., Parker M. W., Cromer B. A. Cytoplasmic ATP-sensing domains regulate gating of skeletal muscle ClC-1 chloride channels. J. Biol. Chem. 2005;280:32452–32458.[PubMed]
20. Niemeyer M. I., Yusef Y. R., Cornejo I., Flores C. A., Sepulveda F. V., Cid L. P. Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies. Physiol. Genomics. 2004;19:74–83.[PubMed]
21. Vanoye C. G., George A. L., Jr Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells. J. Physiol. 2002;539:373–383.
22. Kogan I., Ramjeesingh M., Li C., Bear C. E. Studies of the molecular basis for cystic fibrosis using purified reconstituted CFTR protein. Methods Mol. Med. 2002;70:143–157.[PubMed]
23. Maduke M., Williams C., Miller CFormation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains. Biochemistry. 1998;37:1315–1321.[PubMed][Google Scholar]
24. Li C., Ramjeesingh M., Wang W., Garami E., Hewryk M., Lee D., Rommens J. M., Galley K., Bear C. E. ATPase activity of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 1996;271:28463–28468.[PubMed]
25. Ramjeesingh M., Li C., Garami E., Huan L. J., Galley K., Wang Y., Bear C. E. Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator) Biochemistry. 1999;38:1463–1468.[PubMed]
26. Argos P., Rossman M. G., Grau U. M., Zuber H., Frank G., Tratschin J. D. Thermal stability and protein structure. Biochemistry. 1979;18:5698–5703.[PubMed]
27. Picollo A., Pusch MChloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature (London) 2005;436:420–423.[PubMed][Google Scholar]
28. Scheel O., Zdebik A. A., Lourdel S., Jentsch T. J. Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature (London) 2005;436:424–427.[PubMed]
29. van Weert A. W., Geuze H. J., Groothuis B., Stoorvogel W. Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralisation of endosomal pH nor osmotic swelling of endosomes. Eur. J. Cell Biol. 2000;79:394–399.[PubMed]
30. Wagner C. A., Finberg K. E., Breton S., Marshansky V., Brown D., Geibel J. P. Renal vacuolar H^-ATPase.Physiol. Rev. 2004;84:1263–1314.[PubMed]