CLC proteins and their chloride/proton exchange function

zdebik
Dr Anselm Zdebik
Senior Lecturer in Physiology
Tel: +44 (0)207 794 0500
Email: a.zdebik@ucl.ac.uk

Research

CLC proteins have been known as chloride channels for many years and have been implicated in a variety of diseases (1). KO mice for ClC-2, -3, and ClC-6 have revealed further interesting functions of this protein family (2-4). During my postdoctoral fellowship in Thomas Jentsch’ laboratory (www.fmp-berlin.de/jentsch.html), I have worked on and generated several CLC-related knock-in mouse models. After the discovery of chloride/proton antiport in a bacterial CLC homologue (5), Michael Puschs group (www.ge.cnr.it/ICB/conti_moran_pusch/programs-pusch/home-mik.htm) and ourselves found that ClC-4 and ClC-5 are exchangers rather than chloride channels (6,7). Chloride/proton antiport has recently also been shown for lysosomes (8) expressing almost exclusively ClC-7 and its subunit, Ostm1 (9). As revealed by single channel recordings of concatemeric CLC channels (10) and bacterial crystal structures of CLC exchangers (11), these proteins function as dimers. CLC exchangers can be converted to pure anion conductances by neutralizing a “gating glutamate” conserved in (almost) all CLC proteins. This glutamate will open the chloride pathway when protonated in the wild-type transporter, thereby coupling anion to proton movement, as depicted in Fig. 1.

zdebikfig1

Figure 1.

Current work in the laboratory focuses on understanding chloride/proton exchange in mammalian CLC proteins, using molecular models of mammalian exchangers derived from the bacterial crystal structures and testing hypotheses by site-directed mutagenesis and functional expression in mammalian cells and Xenopus oocytes. In collaboration with Michael Pusch, we have looked at several aspects of ClC-4- and ClC-5-mediated antiport (12): Its anion dependence, its dependence on a certain “proton glutamate” (believed to transfer protons to the “gating glutamate”) and the question whether the transport occurs independently in the two subunits of the dimeric protein. We observed chloride/proton exchange independent of the other subunit in concatemeric constructs linking an exchanging and pure chloride conducting subunit, or an exchanging and a non-functional subunit. A working model for chloride/proton exchange is shown in Fig. 1. Future work will investigate several other aspects of exchange which are still incompletely understood, as well as aspects of CLC channel gating, which may be intricately intertwined with proton movement.

Kidney physiology and genetics of renal disease Further interests include understanding kidney physiology using knock-out models and genetics of renal disease and I am also working on proteins identified by Prof. Robert Kleta in human kidney disease.

Techniques To assess wild-type and mutant channels and transporters we express these proteins in the plasma membrane of mammalian cells for whole cell patch clamp, proton transport measurements and single channel analysis, and use Xenopus oocytes to investigate them for macroscopic currents and proton transport. The latter has been greatly facilitated by a device I developed in the laboratory of Thomas Jentsch coined “Fluorocyte” which allows rapid solution exchange, two-electrode voltage clamp and sensitive semiquantitative pH measurements simultaneously. Mutant proteins are often non-functional. To assure surface expression, we insert HA tags in extracellular loops of these proteins and use surface chemoluminescence in live oocytes. The same oocytes can then be used to assess total protein expression by Western blotting. A PhD position is currently available in the laboratory. The candidate should have a strong interest in biophysics and/or molecular biology and ion transport physiology and should direct inquiries to a.zdebik@ucl.ac.uk.

zdebikfig2

Figure 2.

REFERENCES

  • Jentsch, T. J., Stein, V., Weinreich, F., and Zdebik, A. A. (2002) Physiol Rev 82, 503-568.
  • Bösl, M. R., Stein, V., Hübner, C., Zdebik, A. A., Jordt, S. E., Mukhophadhyay, A. K., Davidoff, M. S., Holstein, A. F., and Jentsch, T. J. (2001) EMBO J 20, 1289-1299
  • Stobrawa, S. M., Breiderhoff, T., Takamori, S., Engel, D., Schweizer, M., Zdebik, A. A., Bösl, M. R., Ruether, K., Jahn, H., Draguhn, A., Jahn, R., and Jentsch, T. J. (2001) Neuron29, 185-196
  • Poet, M., Kornak, U., Schweizer, M., Zdebik, A. A., Scheel, O., Hoelter, S., Wurst, W., Schmitt, A., Fuhrmann, J. C., Planells-Cases, R., Mole, S. E., Hubner, C. A., and Jentsch, T. J. (2006) Proc Natl Acad Sci U S A 103, 13854-13859
  • Accardi, A., and Miller, C. (2004) Nature 427, 803-807
  • Picollo, A., and Pusch, M. (2005) Nature 436, 420-423
  • Scheel, O., Zdebik, A. A., Lourdel, S., and Jentsch, T. J. (2005) Nature 436, 424-427
  • Graves, A. R., Curran, P. K., Smith, C. L., and Mindell, J. A. (2008) Nature
  • Lange, P. F., Wartosch, L., Jentsch, T. J., and Fuhrmann, J. C. (2006) Nature 440, 220-223
  • Weinreich, F., and Jentsch, T. J. (2001) J Biol Chem 276, 2347-2353
  • Dutzler, R., Campbell, E. B., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Nature 415, 287-294.
  • Miller, C. (2006) Nature 440, 484-489
  • Zdebik, A. A., Zifarelli, G., Bergsdorf, E. Y., Soliani, P., Scheel, O., Jentsch, T. J., and Pusch, M. (2008) J Biol Chem 283, 4219-4227
  • Slawik, M., Zdebik, A., Hug, M. J., Kerstan, D., Leipziger, J., and Greger, R. (1996) Pflugers Arch 432, 112-120.
  • Zdebik, A., Hug, M. J., and Greger, R. (1996) Cell Physiol Biochem, 123-128
  • Zdebik, A., Hug, M. J., and Greger, R. (1997) Pflügers Arch 434, 188-194
  • Zdebik, A. A., Cuffe, J. E., Bertog, M., Korbmacher, C., and Jentsch, T. J. (2004) J Biol Chem 279, 22276-22283
  • Jentsch, T. J., Maritzen, T., and Zdebik, A. A. (2005) J Clin Invest 115, 2039-2046
  • Jentsch, T. J., Poet, M., Fuhrmann, J. C., and Zdebik, A. A. (2005) Annu Rev Physiol 67, 779-807

PAST RESEARCH

  • Characterization of whole-cell conductances in rat pancretic acini at rest and during calcium agonist stimulation (20, 21)
  • Investigation of the secretory calcium-activated chloride channel in these cells at the single-channel level (22)
  • Establishment of Ussing chamber measurements for mouse retinal pigment epithelium and investigation of the effect of ClC-2 KO on its properties (24)
  • Characterization of volume-activated currents in pancreatic acini and hepatocytes Characterization of volume-activated currents in pancreatic acini and hepatocytes
  • Ussing chamber measurements on ClC-2 KO, CFTR KO, and CFTR/CLC-2 double KO colon to shed light on ClC-2 as a putative CFTR rescue channel (27)
  • Generation of conditional barttin KO mice to dissect the role of ClC-K/barttin channels in different parts of the nephron (manuscript in preparation)
  • Structure-function studies on the chloride/proton transport activity of mammalian CLC proteins (28, 30), ongoing

REVIEWS

  • Jentsch TJ, Maritzen T, and Zdebik AA. (2005) Chloride channel diseases resulting from impaired transepithelial transport or vesicular function. J Clin Invest 115: 2039-2046
  • Jentsch TJ, Poët M, Fuhrmann JC, and Zdebik AA. (2005) Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67: 779-807
  • Jentsch TJ, Stein V, Weinreich F, and Zdebik AA. (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503-568, 2002.


Full list of publications with PDF links

SELECTED PUBLICATIONS

  • Slawik M, Zdebik A, Hug MJ, Kerstan D, Leipziger J, and Greger R. (1996) Whole-cell conductive properties of rat pancreatic acini.
  • Pflügers Arch 432: 112-120
  • Zdebik A, Hug MJ, and Greger R. (1996) Low concentrations of carbachol induce oscillations of membrane voltage in rat pancreatic acinar cells - no evidencefor the activation of non-selective cation channels. Cell Physiol Biochem 6: 123-128
  • Zdebik A, Hug MJ, and Greger R. (1997) Chloride channels in the luminal membrane of rat pancreatic acini. Pflügers Arch 434: 188-194
  • Schumacher YO, Zdebik A, Huonker M, and Kreisel W. (2001) Sildenafil in HIV-related pulmonary hypertension. Aids 15: 1747-1748
  • Bösl MR*, Stein V*, Hübner C, Zdebik AA, Jordt SE, Mukhophadhyay AK, Davidoff MS, Holstein AF, and Jentsch TJ. (2001) Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl--channel disruption. Neuron 29, 185-196
  • Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bösl MR, Ruether K, Jahn H, Draguhn A, Jahn R, and Jentsch TJ. (2001) Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29: 185-196, 2001
  • Warth R, Garcia Alzamora M, Kim JK, Zdebik A, Nitschke R, Bleich M, Gerlach U, Barhanin J, and Kim SJ. (2002) The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse. Pflügers Arch 443: 822-828
  • Jentsch TJ, Stein V, Weinreich F, and Zdebik AA. (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503-568, 2002.
  • Zdebik AA, Cuffe JE, Bertog M, Korbmacher C, and Jentsch TJ (2004) Additional disruption of the ClC-2 Cl(-) channel does not exacerbate the cystic fibrosis phenotype of cystic fibrosis transmembrane conductance regulator mouse models. J Biol Chem 279: 22276-22283
  • Scheel O*, Zdebik AA*, Lourdel S, and Jentsch TJ. (2005) Voltage-dependent electro-genic chloride/proton exchange by endosomal CLC proteins. Nature 436: 424-427
  • Jentsch TJ, Maritzen T, and Zdebik AA. (2005) Chloride channel diseases resulting from impaired transepithelial transport or vesicular function. J Clin Invest 115: 2039-2046
  • Jentsch TJ, Poët M, Fuhrmann JC, and Zdebik AA. (2005) Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67: 779-807
  • Poet M, Kornak U, Schweizer M, Zdebik AA, Scheel O, Hoelter S, Wurst W, Schmitt A, Fuhrmann JC, Planells-Cases R, Mole SE, Hübner CA, and Jentsch TJ. (2006) Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Acad Sci U S A 103: 13854-13859
  • Zdebik AA*, Zifarelli G*, Bergsdorf E-Y, Soliani P, Scheel O, Jentsch TJ, and Pusch M. (2007) Determinants of anion-proton coupling in mammalian endosomal CLC proteins. J Biol Chem