Abstract
The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.
Full Text
The Full Text of this article is available as a PDF (207.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amann K. J., Guo A. W., Ervasti J. M. Utrophin lacks the rod domain actin binding activity of dystrophin. J Biol Chem. 1999 Dec 10;274(50):35375–35380. doi: 10.1074/jbc.274.50.35375. [DOI] [PubMed] [Google Scholar]
- Cantiello H. F., Stow J. L., Prat A. G., Ausiello D. A. Actin filaments regulate epithelial Na+ channel activity. Am J Physiol. 1991 Nov;261(5 Pt 1):C882–C888. doi: 10.1152/ajpcell.1991.261.5.C882. [DOI] [PubMed] [Google Scholar]
- Cid L. P., Montrose-Rafizadeh C., Smith D. I., Guggino W. B., Cutting G. R. Cloning of a putative human voltage-gated chloride channel (CIC-2) cDNA widely expressed in human tissues. Hum Mol Genet. 1995 Mar;4(3):407–413. doi: 10.1093/hmg/4.3.407. [DOI] [PubMed] [Google Scholar]
- Downey G. P., Grinstein S., Sue-A-Quan A., Czaban B., Chan C. K. Volume regulation in leukocytes: requirement for an intact cytoskeleton. J Cell Physiol. 1995 Apr;163(1):96–104. doi: 10.1002/jcp.1041630111. [DOI] [PubMed] [Google Scholar]
- Duan D., Ye L., Britton F., Horowitz B., Hume J. R. A novel anionic inward rectifier in native cardiac myocytes. Circ Res. 2000 Mar 3;86(4):E63–E71. [PubMed] [Google Scholar]
- Foskett J. K., Spring K. R. Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation. Am J Physiol. 1985 Jan;248(1 Pt 1):C27–C36. doi: 10.1152/ajpcell.1985.248.1.C27. [DOI] [PubMed] [Google Scholar]
- Gründer S., Thiemann A., Pusch M., Jentsch T. J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. Nature. 1992 Dec 24;360(6406):759–762. doi: 10.1038/360759a0. [DOI] [PubMed] [Google Scholar]
- Higashi T., Richards C. S., Uyeda K. The interaction of phosphofructokinase with erythrocyte membranes. J Biol Chem. 1979 Oct 10;254(19):9542–9550. [PubMed] [Google Scholar]
- Jentsch T. J., Friedrich T., Schriever A., Yamada H. The CLC chloride channel family. Pflugers Arch. 1999 May;437(6):783–795. doi: 10.1007/s004240050847. [DOI] [PubMed] [Google Scholar]
- Jordt S. E., Jentsch T. J. Molecular dissection of gating in the ClC-2 chloride channel. EMBO J. 1997 Apr 1;16(7):1582–1592. doi: 10.1093/emboj/16.7.1582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kurashima K., D'Souza S., Szászi K., Ramjeesingh R., Orlowski J., Grinstein S. The apical Na(+)/H(+) exchanger isoform NHE3 is regulated by the actin cytoskeleton. J Biol Chem. 1999 Oct 15;274(42):29843–29849. doi: 10.1074/jbc.274.42.29843. [DOI] [PubMed] [Google Scholar]
- Levitan I., Almonte C., Mollard P., Garber S. S. Modulation of a volume-regulated chloride current by F-actin. J Membr Biol. 1995 Oct;147(3):283–294. doi: 10.1007/BF00234526. [DOI] [PubMed] [Google Scholar]
- Malinowska D. H., Kupert E. Y., Bahinski A., Sherry A. M., Cuppoletti J. Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel. Am J Physiol. 1995 Jan;268(1 Pt 1):C191–C200. doi: 10.1152/ajpcell.1995.268.1.C191. [DOI] [PubMed] [Google Scholar]
- Matthews J. B., Smith J. A., Hrnjez B. J. Effects of F-actin stabilization or disassembly on epithelial Cl- secretion and Na-K-2Cl cotransport. Am J Physiol. 1997 Jan;272(1 Pt 1):C254–C262. doi: 10.1152/ajpcell.1997.272.1.C254. [DOI] [PubMed] [Google Scholar]
- Murthy S. N., Liu T., Kaul R. K., Köhler H., Steck T. L. The aldolase-binding site of the human erythrocyte membrane is at the NH2 terminus of band 3. J Biol Chem. 1981 Nov 10;256(21):11203–11208. [PubMed] [Google Scholar]
- Park K., Arreola J., Begenisich T., Melvin J. E. Comparison of voltage-activated Cl- channels in rat parotid acinar cells with ClC-2 in a mammalian expression system. J Membr Biol. 1998 May 15;163(2):87–95. doi: 10.1007/s002329900373. [DOI] [PubMed] [Google Scholar]
- Pasyk E. A., Morin X. K., Zeman P., Garami E., Galley K., Huan L. J., Wang Y., Bear C. E. A conserved region of the R domain of cystic fibrosis transmembrane conductance regulator is important in processing and function. J Biol Chem. 1998 Nov 27;273(48):31759–31764. doi: 10.1074/jbc.273.48.31759. [DOI] [PubMed] [Google Scholar]
- Pusch M., Jordt S. E., Stein V., Jentsch T. J. Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol. 1999 Mar 1;515(Pt 2):341–353. doi: 10.1111/j.1469-7793.1999.341ac.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwiebert E. M., Cid-Soto L. P., Stafford D., Carter M., Blaisdell C. J., Zeitlin P. L., Guggino W. B., Cutting G. R. Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3879–3884. doi: 10.1073/pnas.95.7.3879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwiebert E. M., Mills J. W., Stanton B. A. Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line. J Biol Chem. 1994 Mar 11;269(10):7081–7089. [PubMed] [Google Scholar]
- Spector I., Shochet N. R., Blasberger D., Kashman Y. Latrunculins--novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D. Cell Motil Cytoskeleton. 1989;13(3):127–144. doi: 10.1002/cm.970130302. [DOI] [PubMed] [Google Scholar]
- Staley K., Smith R., Schaack J., Wilcox C., Jentsch T. J. Alteration of GABAA receptor function following gene transfer of the CLC-2 chloride channel. Neuron. 1996 Sep;17(3):543–551. doi: 10.1016/s0896-6273(00)80186-5. [DOI] [PubMed] [Google Scholar]
- Thiemann A., Gründer S., Pusch M., Jentsch T. J. A chloride channel widely expressed in epithelial and non-epithelial cells. Nature. 1992 Mar 5;356(6364):57–60. doi: 10.1038/356057a0. [DOI] [PubMed] [Google Scholar]
- Tilly B. C., Edixhoven M. J., Tertoolen L. G., Morii N., Saitoh Y., Narumiya S., de Jonge H. R. Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton. Mol Biol Cell. 1996 Sep;7(9):1419–1427. doi: 10.1091/mbc.7.9.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vince J. W., Reithmeier R. A. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1-/HCO3- exchanger. J Biol Chem. 1998 Oct 23;273(43):28430–28437. doi: 10.1074/jbc.273.43.28430. [DOI] [PubMed] [Google Scholar]
- Yu J., Steck T. L. Isolation and characterization of band 3, the predominant polypeptide of the human erythrocyte membrane. J Biol Chem. 1975 Dec 10;250(23):9170–9175. [PubMed] [Google Scholar]