Transmembrane topology of a CLC chloride channel

T Schmidt-Rose

T Jentsch

Center for Molecular Neurobiology Hamburg, ZMNH, Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany

^{To whom reprint requests should be addressed.}

William A. Catterall, University of Washington School of Medicine, Seattle, WA

Received 1997 Feb 13; Accepted 1997 May 12.

Abstract

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure–function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of in vitro glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.

Abstract

Voltage-gated chloride channels of the CLC family are highly conserved during evolution and are expressed in organisms ranging from bacteria (1) and yeast (2) to plants (3) and animals (4). Their physiological functions in higher organisms include the regulation of cell volume, control of electrical excitability, and transepithelial transport.

The CLC proteins were identified by expression cloning of the Torpedo chloride channel ClC-0 (5). At present, nine mammalian members are known (for review, see ref. 6). ClC-1 is nearly specific for skeletal muscle (4). It ensures its electrical stability, which is evident from ClC-1 mutations leading to myotonia congenita, a disease characterized by defective muscle relaxation. Many different ClC-1 mutations were found in human myotonia and in animal models (7–11). The importance of CLC channels is underscored by another, recently identified disease. Inactivating mutations of ClC-5 cause a syndrome that is characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones (12). ClC-2 may play a role in cell volume control (13) and in setting the intracellular chloride concentration, which in turn is important for synaptic transmission in certain neurons (14). However, the function of most CLC proteins is still unclear.

CLC proteins are structurally unrelated to other ion channel classes. Topological models are still mainly based on hydropathy analysis, suggesting the presence of about 12–13 transmembrane domains, originally termed D1 to D13 (5). This model has already had to be revised to accommodate new experimental findings (15, 16).

CLC channels function as multimers of identical (11) or homologous subunits (17). Dominant negative mutations suggested that ClC-1 channels have more than three subunits (11). However, ClC-0 is a homodimeric channel with one pore per subunit (16, 18, 19); this may also apply for ClC-1 (20).

In structure–function studies, mutations have been introduced into ClC-0 (18, 21), ClC-1 (9, 11), ClC-2 (13, 22), and ClC-5 (12). These mutations showed that several protein regions are important for gating and permeation. Unfortunately, the interpretation of these data is limited by the lack of structural information.

We put the topology of CLC channels on a firm experimental basis. We primarily used the glycosylation scanning procedure (23–25), which we complemented with protease protection assays (26, 27) and by introducing cysteines that were then probed with extracellular cysteine-modifying reagents for effects on currents (28–30). In addition to clarifying the transmembrane topology, the latter technique identified novel ClC-1 residues, which are important for channel function.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft, the Muscular Dystrophy Association, and the Fonds der Chemischen Industrie.

Acknowledgments

ABBREVIATIONS

MTSES	sodium (2-sulfonatoethyl)methanethiosulfonate
WT	wild type

ABBREVIATIONS

References

1. Fujita N, Mori H, Yura T, Ishihama A. Nucleic Acids Res. 1994;22:1637–1639.
2. Greene J R, Brown N H, DiDomenico B J, Kaplan J, Eide D J. Mol Gen Genet. 1993;241:542–553.[PubMed]
3. Hechenberger M, Schwappach B, Fischer W N, Frommer W B, Jentsch T J, Steinmeyer K. J Biol Chem. 1996;271:33632–33638.[PubMed]
4. Steinmeyer K, Ortland C, Jentsch T J. Nature (London) 1991;354:301–304.[PubMed]
5. Jentsch T J, Steinmeyer K, Schwarz G. Nature (London) 1990;348:510–514.[PubMed]
6. Jentsch T J, Günther W, Pusch M, Schwappach B. J Physiol (London) 1995;482P:19S–26S.
7. George A L, Crackower M A, Abdalla J A, Hudson A J, Ebers G C. Nat Genet. 1993;3:305–309.[PubMed]
8. Koch M C, Steinmeyer K, Lorenz C, Ricker K, Wolf F, Otto M, Zoll B, Lehmann Horn F, Grzeschik K H, Jentsch T J. Science. 1992;257:797–800.[PubMed]
9. Pusch M, Steinmeyer K, Koch M C, Jentsch T J. Neuron. 1995;15:1–20.[PubMed]
10. Steinmeyer K, Klocke R, Ortland C, Gronemeier M, Jockusch H, Gründer S, Jentsch T J. Nature (London) 1991;354:304–308.[PubMed]
11. Steinmeyer K, Lorenz C, Pusch M, Koch M C, Jentsch T J. EMBO J. 1994;13:737–743.
12. Lloyd S E, Pearce S H, Fisher S E, Steinmeyer K, Schwappach B, Scheinman S J, Harding B, Bolino A, Devoto M, Goodyer P, Rigden S P, Wrong O, Jentsch T J, Craig I W, Thakker R V. Nature (London) 1996;379:445–449.[PubMed]
13. Gründer S, Thiemann A, Pusch M, Jentsch T J. Nature (London) 1992;360:759–762.[PubMed]
14. Staley K J, Smith R, Schaak J, Wilcox C, Jentsch T J. Neuron. 1996;17:543–551.[PubMed]
15. Kieferle S, Fong P, Bens M, Vandewalle A, Jentsch T J. Proc Natl Acad Sci USA. 1994;91:6943–6947.
16. Middleton R E, Pheasant D J, Miller C. Biochemistry. 1994;33:13189–13198.[PubMed]
17. Lorenz C, Pusch M, Jentsch T J. Proc Natl Acad Sci USA. 1996;93:13362–13366.
18. Ludewig U, Pusch M, Jentsch T J. Nature (London) 1996;383:340–343.[PubMed]
19. Middleton R E, Pheasant D J, Miller C. Nature (London) 1996;383:337–340.[PubMed]
20. Fahlke C, Knittel T, Gurnett C A, Campbell K P, George A L. J Gen Physiol. 1997;109:93–104.
21. Pusch M, Ludewig U, Rehfeldt A, Jentsch T J. Nature (London) 1995;373:527–531.[PubMed]
22. Jordt S E, Jentsch T J. EMBO J. 1997;16:1582–1592.
23. Chavez R A, Hall Z W. J Biol Chem. 1991;266:15532–15538.[PubMed]
24. Chang X B, Hou Y X, Jensen T J, Riordan J R. J Biol Chem. 1994;269:18572–18575.[PubMed]
25. Turk E, Kerner C J, Lostao M P, Wright E M. J Biol Chem. 1996;271:1925–1934.[PubMed]
26. Chavez R A, Hall Z W. J Cell Biol. 1992;116:385–393.
27. Skach W R, Lingappa V R. Cancer Res. 1994;54:3202–3209.[PubMed]
28. Akabas M H, Kaufmann C, Cook T A, Archdeacon P. J Biol Chem. 1994;269:14865–14868.[PubMed]
29. Kürz L L, Zühlke R D, Zhang H J, Joho R H. Biophys J. 1995;68:900–905.
30. Perez-Garcia M T, Chiamvimonvat N, Marban E, Tomaselli G F. Proc Natl Acad Sci USA. 1996;93:300–304.
31. Higuchi R In: PCR Protocols. Innis M A, Gelfand D H, Sinnsky J J, White T J, editors. San Diego: Academic; 1990. pp. 177–183. [PubMed][Google Scholar]
32. Nilsson I M, von Heijne G. J Biol Chem. 1993;268:5798–5801.[PubMed]
33. Landolt-Marticorena C, Reithmeier R A. Biochem J. 1994;302:253–260.
34. Hresko R C, Kruse M, Strube M, Mueckler M. J Biol Chem. 1994;269:20482–20488.[PubMed]
35. Perara E, Lingappa V R. J Cell Biol. 1985;101:2292–2301.
36. Rothman R E, Andrews D W, Calayag M C, Lingappa V R. J Biol Chem. 1988;263:10470–10480.[PubMed]
37. Skach W R, Lingappa V R. J Biol Chem. 1993;268:23552–23561.[PubMed]
38. Akabas M H, Stauffer D A, Xu M, Karlin A. Science. 1992;258:307–310.[PubMed]
39. Kuner T, Wollmuth L P, Karlin A, Seeburg P H, Sakmann B. Neuron. 1996;17:343–352.[PubMed]
40. Pascual J M, Shieh C C, Kirsch G E, Brown A M. Neuron. 1995;14:1055–1063.[PubMed]
41. Akabas M H, Karlin A. Biochemistry. 1995;34:12496–12500.[PubMed]
42. Cheung M, Akabas M H. Biophys J. 1996;70:2688–2695.
43. Loo T W, Clarke D M. J Biol Chem. 1995;270:843–848.[PubMed]
44. Traxler B, Boyd D, Beckwith J. J Membr Biol. 1993;132:1–11.[PubMed]
45. Kyte J, Doolittle R F. J Mol Biol. 1982;157:105–132.[PubMed]
46. von Heijne G, Gavel Y. Eur J Biochem. 1988;174:671–678.[PubMed]
47. Sipos L, von Heijne G. Eur J Biochem. 1993;213:1333–40.[PubMed]
48. Skach W R, Shi L B, Calayag M C, Frigeri A, Lingappa V R, Verkman A S. J Cell Biol. 1994;125:803–815.
49. Henn D K, Baumann A, Kaupp U B. Proc Natl Acad Sci USA. 1995;92:7425–7429.
50. Lin J, Addison R. J Biol Chem. 1995;270:6935–6941.[PubMed]
51. Jentsch T J, Günther W, Pusch M, Schwappach B. J Physiol (London) 1995;482:19S–25S.
52. Persson B, Argos P. J Mol Biol. 1994;237:182–192.[PubMed]
53. Rost B, Casadio R, Fariselli P, Sander C. Protein Sci. 1995;4:521–533.
54. Rost B. Methods Enzymol. 1996;266:525–539.[PubMed]
55. Rost B, Fariselli B, Casadio R. Protein Sci. 1996;7:1704–1718.
56. Adachi S, Uchida S, Ito H, Hata M, Hiroe M, Marumo F, Sasaki S. J Biol Chem. 1994;269:17677–17683.[PubMed]
57. Zimniak L, Winters C J, Reeves W B, Andreoli T E. Am J Physiol. 1996;270:F1066–F1072.[PubMed]