Cell and Tissue Biology

, Volume 6, Issue 1, pp 52–59 | Cite as

Mechanisms of epithelial sodium channel (ENaC) regulation by cortactin: Involvement of dynamin

  • D. V. Ilatovskaya
  • T. S. Pavlov
  • Yu. A. Negulyaev
  • A. Staruschenko


We have recently shown that epithelial sodium channels (ENaCs) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. It has been also demonstrated that a GTPase dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. Thus, treatment of mpkCCDc14 cells monolayers with dynasore (in concentrations of 10 and 100 μM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, dynasore caused reduced amiloride-sensitive currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel’s activity and use of this drug is not appropriate for studies of ENaC endocytosis. We hypothesize that this effect is most likely caused either by dynasore’s toxic actions upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin (K44A) transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC only (control); additional transfection of cortactin together with the K44A dynamin resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease of ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but also through the dynamin-mediated processes.


ENaC cortactin dynamin endocytosis dynasore sodium transport epithelial cells 


  1. Alvarez de la Rosa, D., Canessa, C.M., Fyfe, G.K., and Zhang, P., Structure and Regulation of Amiloride-sensitive Sodium Channels, Annu. Rev. Physiol., 2000, vol. 62, pp. 573–594.PubMedCrossRefGoogle Scholar
  2. Bens, M., Vallet, V., Cluzeaud, F., Pascual-Letallec, L., Kahn, A., Rafestin-Oblin, M.E., Rossier, B.C., and Vandewalle, A., Corticosteroid-Dependent Sodium Transport in a Novel Immortalized Mouse Collecting duct Principal Cell Line, J. Am. Soc. Nephrol., 1999, vol. 10, pp. 923–934.PubMedGoogle Scholar
  3. Blazer-Yost, B.L., Esterman, M.A., and Vlahos, C.J., Insulin-Stimulated Trafficking of ENaC in Renal Cells Requires PI 3-Kinase Activity, Am. J. Physiol. Cell Physiol., 2003, vol. 284, pp. C1645–C1653.PubMedGoogle Scholar
  4. Campbell, D.H., Sutherland, R.L., and Daly, R.J., Signaling Pathways and Structural Domains Required for Phosphorylation of EMS1/Cortactin, Cancer Res., 1999, vol. 59, pp. 5376–5385.PubMedGoogle Scholar
  5. Cantiello, H.F., Stow, J.L., Prat, A.G., and Ausiello, D.A., Actin Filaments Regulate Epithelial Na+ Channel Activity, Am. J. Physiol., 1991, vol. 261, pp. C882–C888.PubMedGoogle Scholar
  6. Cao, H., Thompson, H.M., Krueger, E.W., and McNiven, M.A., Disruption of Golgi Structure and Function in Mammalian Cells Expressing a Mutant Dynamin, J. Cell Sci., 2000, vol. 113, pp. 1993–2002.PubMedGoogle Scholar
  7. Chang, S.S., Grunder, S., Hanukoglu, A., Rosler, A., Mathew, P.M., Hanukoglu, I., Schild, L., Lu, Y., Shimkets, R.A., Nelson-Williams, C., Rossier, B.C., and Lifton, R.P., Mutations in Subunits of the Epithelial Sodium Channel Cause Salt Wasting with Hyperkalaemic Acidosis, Pseudohypoaldosteronism Type 1, Nat. Genet., 1996, vol. 12, pp. 248–253.PubMedCrossRefGoogle Scholar
  8. Cosen-Binker, L.I. and Kapus, A., Cortactin: The Gray Eminence of the Cytoskeleton, Physiology (Bethesda), 2006, vol. 21, pp. 352–361.CrossRefGoogle Scholar
  9. Gallet, C., Rosa, J.P., Habib, A., Lebret, M., Levy-Toledano, S., and Maclouf, J., Tyrosine Phosphorylation of Cortactin Associated with Syk Accompanies Thromboxane Analogue-Induced Platelet Shape Change, J. Biol. Chem., 1999, vol. 274, pp. 23610–23616.PubMedCrossRefGoogle Scholar
  10. Garty, H. and Palmer, L.G., Epithelial Sodium Channels: Function, Structure, and Regulation, Physiol. Rev., 1997, vol. 77, pp. 359–396.PubMedGoogle Scholar
  11. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J., Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches, Pflügers Archiv, 1981, vol. 391, pp. 85–100.PubMedCrossRefGoogle Scholar
  12. Hansson, J.H., Nelson-Williams, C., Suzuki, H., Schild, L., Shimkets, R., Lu, Y., Canessa, C., Iwasaki, T., Rossier, B, and Lifton, R.P., Hypertension Caused by a Truncated Epithelial Sodium Channel Gamma Subunit: Genetic Heterogeneity of Liddle Syndrome, Nat. Genet., 1995, vol. 11, pp. 76–82.PubMedCrossRefGoogle Scholar
  13. Huang, C., Ni, Y., Wang, T., Gao, Y., Haudenschild, C.C., and Zhan, X., Down-Regulation of the Filamentous Actin Cross-Linking Activity of Cortactin by Src-Mediated Tyrosine Phosphorylation, J. Biol. Chem., 1997, vol. 272, pp. 13911–13915.PubMedCrossRefGoogle Scholar
  14. Ilatovskaya, D.V., Levchenko, V., Ryan, R.P., Cowley, A.W., Jr., and Staruschenko, A.A., NSAIDs Acutely Inhibit TRPC Channels in Freshly Isolated Rat Glomeruli, Bichem. Biophys. Res. Commun., 2011a, vol. 408, pp. 242–247.CrossRefGoogle Scholar
  15. Ilatovskaya, D.V., Pavlov, T.S., Levchenko, V., Negulyaev, Y.A., and Staruschenko, A.A., Cortical Actin Binding Protein Cortactin Mediates ENaC Activity via Arp2/3 Complex, FASEB J., 2011b, vol. 25, pp. 2688–2699.PubMedCrossRefGoogle Scholar
  16. Kanner, S.B., Reynolds, A.B., Vines, R.R., and Parsons, J.T., Monoclonal Antibodies to Individual Tyrosine-Phosphorylated Protein Substrates of Oncogene-Encoded Tyrosine Kinases, Proc. Natl. Acad. Sci. USA, 1990, vol. 87, pp. 3328–3332.PubMedCrossRefGoogle Scholar
  17. Kapus, A., Szaszi, K., Sun, J., Rizoli, S., and Rotstein, O.D., Cell Shrinkage Regulates Src Kinases and Induces Tyrosine Phosphorylation of Cortactin, Independent of the Osmotic Regulation of Na+/H+ Exchangers, J. Biol. Chem., 1999, vol. 274, pp. 8093–8102.PubMedCrossRefGoogle Scholar
  18. Kapus, A., Di, Ciano, C., Sun, J., Zhan, X., Kim, L., Wong, T.W., and Rotstein, O.D., Cell Volume-Dependent Phosphorylation of Proteins of the Cortical Cytoskeleton and Cell-Cell Contact Sites. The Role of Fyn and FER Kinases, J. Biol. Chem., 2000, vol. 275, pp. 32289–32298.PubMedCrossRefGoogle Scholar
  19. Karpushev, A.V., Levchenko, V., Pavlov, T.S., Lam, V.Y., Vinnakota, K.C., Vandewalle, A., Wakatsuki, T., and Staruschenko, A., Regulation of ENaC Expression at the Cell Surface by Rab11, Biochem. Biophys. Res. Commun., 2008, vol. 377, pp. 521–525.PubMedCrossRefGoogle Scholar
  20. Karpushev, A.V., Ilatovskaya, D.V., Pavlov, T.S., Negulyaev, Y.A., and Staruschenko, A.A., Intact Cytoskeleton Is Required for Small G Protein Dependent Activation of the Epithelial Na+ Channel, PLoS One, 2010a, vol. 5, p. e8827.PubMedCrossRefGoogle Scholar
  21. Karpushev, A.V., Ilatovskaya, D.V., and Staruschenko, A.B., The Actin Cytoskeleton and Small G Protein RhoA Are not Involved in Flow-Dependent Activation of ENaC, BMC Res. Notes, 2010b, vol. 3, p. 210.PubMedCrossRefGoogle Scholar
  22. Karpushev, A.V., Levchenko, V., Ilatovskaya, D.V., Pavlov, T.S., and Staruschenko, A., Novel Role of Rac1/WAVE Signaling Mechanism in Regulation of the Epithelial Na+ Channel, Hypertension, 2011, vol. 57, pp. 996–1002.PubMedCrossRefGoogle Scholar
  23. Kellenberger, S. and Schild, L., Epithelial Sodium Channel/Degenerin Family of Ion Channels: A Variety of Functions for a Shared Structure, Physiol. Rev., 2002, vol. 82, pp. 735–767.PubMedGoogle Scholar
  24. Kirchhausen, T., Macia, E., and Pelish, H.E., Use of Dynasore, the Small Molecule Inhibitor of Dynamin, in the Regulation of Endocytosis, Methods Enzymol., 2008, vol. 438, pp. 77–93.PubMedCrossRefGoogle Scholar
  25. Levchenko, V., Zheleznova, N.N., Pavlov, T.S., Vandewalle, A., Wilson, P.D., and Staruschenko, A., EGF and Its Related Growth Factors Mediate Sodium Transport in MpkCCD(c14) Cells via ErbB2 (neu/HER-2) Receptor, J. Cell. Physiol., 2010, vol. 223, pp. 252–259.PubMedGoogle Scholar
  26. Lifton, R.P., Genetic Determinants of Human Hypertension, Proc. Natl. Acad. Sci. USA, 1995, vol. 92, pp. 8545–8551.PubMedCrossRefGoogle Scholar
  27. Martinez-Quiles, N., Ho, H.Y., Kirschner, M.W., Ramesh, N., and Geha, R.S., Erk/Src Phosphorylation of Cortactin Acts as a Switch On/Switch Off Mechanism That Controls Its Ability to Activate N-WASP, Mol. Cell Biol., 2004, vol. 24, pp. 5269–5280.PubMedCrossRefGoogle Scholar
  28. Mazzochi, C., Benos, D.J., and Smith, P.R., Interaction of Epithelial Ion Channels with the Actin-Based Cytoskeleton, Am. J. Physiol. Renal Physiol., 2006a, vol. 291, pp. F1113–F1122.PubMedCrossRefGoogle Scholar
  29. Mazzochi, C., Bubien, J.K., Smith, P.R., and Benos, D.J., The Carboxyl Terminus of the Alpha-Subunit of the Amiloride-Sensitive Epithelial Sodium Channel Binds to F-Actin, J. Biol. Chem., 2006b, vol. 281, pp. 6528–6538.PubMedCrossRefGoogle Scholar
  30. Meighan, S.E., Meighan, P.C., Choudhury, P., Davis, C.J., Olson, M.L., Zornes, P.A., Wright, J.W., and Harding, J.W., Effects of Extracellular Matrix-Degrading Proteases Matrix Metalloproteinases 3 and 9 on Spatial Learning and Synaptic Plasticity, J. Neurochem., 2006, vol. 96, pp. 1227–1241.PubMedCrossRefGoogle Scholar
  31. Mettlen, M., Pucaduil, T., Ramachandran, R., and Schmid, S.L., Dissecting Dynamin’s Role in Clathrin-Mediated Endocytosis, Biochem. Soc. Trans., 2009, vol. 37, pp. 1022–1026.PubMedCrossRefGoogle Scholar
  32. Mizutani, K., Miki, H., He, H., Maruta, H., and Takenawa, T., Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Podosome Formation and Degradation of Extracellular Matrix in Src-Transformed Fibroblasts, Cancer Res., 2002, vol. 62, pp. 669–674.PubMedGoogle Scholar
  33. Mooren, O.L., Kotova, T.I., Moore, A.J., and Schafer, D.A., Dynamin2 GTPase and Cortactin Remodel Actin Filaments, J. Biol. Chem., 2009, vol. 284, pp. 23995–24005.PubMedCrossRefGoogle Scholar
  34. Rossier, B.C. and Shield, R., Epithelial Sodium Channel: Mendelian versus Essential Hypertension, Hypertension, 2008, vol. 52, pp. 595–600.PubMedCrossRefGoogle Scholar
  35. Schafer, D.A., Weed, S.A., Binns, D., Karginov, A.V., Parsons, J.T., and Cooper, J.A., Dynamin2 and Cortactin Regulate Actin Assembly and Filament Organization, Curr. Biol., 2002, vol. 12, pp. 1852–1857.PubMedCrossRefGoogle Scholar
  36. Schild, L., The Epithelial Sodium Channel: From Molecule to Disease, Rev. Physiol. Biochem. Pharmacol., 2004, vol. 151, pp. 93–107.PubMedCrossRefGoogle Scholar
  37. Shimkets, R.A., Lifton, R.P., and Canessa, C.M., The Activity of the Epithelial Sodium Channel Is Regulated by Clathrin-Mediated Endocytosis, J. Biol. Chem., 1997, vol. 272, pp. 25537–25541.PubMedCrossRefGoogle Scholar
  38. Staruschenko, A., Medina, J.L., Patel, P., Shapiro, M.S., Booth, R.E., and Stockand, J.D., Fluorescence Resonance Energy Transfer Analysis of Subunit Stoichiometry of the Epithelial Na+ Channel, J. Biol. Chem., 2004a, vol. 279, pp. 27729–27734.PubMedCrossRefGoogle Scholar
  39. Staruschenko, A., Patel, P., Tong, Q., Medina, J.L., and Stockand, J.D., Ras Activates the Epithelial Na(+) Channel through Phosphoinositide 3-OH Kinase Signaling, J. Biol. Chem., 2004b, vol. 279, pp. 37771–37778.PubMedCrossRefGoogle Scholar
  40. Staruschenko, A., Pochynyuk, O.M., and Stockand, J.D., Regulation of Epithelial Na+ Channel Activity by Conserved Serine/Threonine Switches within Sorting Signals, J. Biol. Chem., 2005, vol. 280, pp. 39161–39167.PubMedCrossRefGoogle Scholar
  41. Takai, Y., Sasaki, T., and Maozaki, T., Small GTP-Binding Proteins, Physiol. Rev., 2001, vol. 81, pp. 153–208.PubMedGoogle Scholar
  42. Tian, L., Chen, L., McClafferty, H., Sailer, C.A., Ruth, P., Knaus, H.G., and Shipston, M.J., A Noncanonical SH3 Domain Binding Motif Links BK Channels to the Actin Cytoskeleton via the SH3 Adapter Cortactin, FASEB J., 2006, vol. 20, pp. 2588–2590.PubMedCrossRefGoogle Scholar
  43. Tian, L., McClafferty, H., Chen, L., and Shipston, M.J., Reversible Tyrosine Protein Phosphorylation Regulates Large Conductance Voltage- and Calcium-Activated Potassium Channels via Cortactin, J. Biol. Chem., 2008, vol. 283, pp. 3067–3076.PubMedCrossRefGoogle Scholar
  44. Uruno, T., Liu, J., Zhang, P., Fan, Y., Egile, C., Li, R., Mueller, S.C., and Zhan, X., Activation of Arp2/3 Complex-Mediated Actin Polymerization by Cortactin, Nat. Cell Biol., 2001, vol. 3, pp. 259–266.PubMedCrossRefGoogle Scholar
  45. Vachugova, D.V. and Morachevskaya, E.A., Mechanosensitivity of Cationic Channels of DEG/ENaC Family, Tsitologiia, 2009, vol. 51, no. 10, pp. 806–814.PubMedGoogle Scholar
  46. Vidal, C., Geny, B., Melle, J., Jandrot-Perrus, M., and Fontenay-Roupie, M., Cdc42/Rac1-Dependent Activation of the P21-Activated Kinase (PAK) Regulates Human Platelet Lamellipodia Spreading: Implication of the Cortical-Actin Binding Protein Cortactin, Blood, 2002, vol. 100, pp. 4462–4469.PubMedCrossRefGoogle Scholar
  47. Wang, H., Traub, L.M., Weixel, K.M., Hawryluk, M.J., Shah, N., Edinger, R.S., Perry, C.J., Kester, L., Butterworth, M.B., Peters, K.W., Kleyman, T.R., Frizzell, R.A., and Johnson, J.P., Clathrin-Mediated Endocytosis of the Epithelial Sodium Channel. Role of Epsin, J. Biol. Chem., 2006, vol. 281, pp. 14129–14135.PubMedCrossRefGoogle Scholar
  48. Weed, S.A. and Parsons, J.T., Cortactin: Coupling Membrane Dynamics to Cortical Actin Assembly, Oncogene, 2001, vol. 20, pp. 6418–6434.PubMedCrossRefGoogle Scholar
  49. Williams, M.R., Markey, J.C., Doczi, M.A., and Morielli, A.D., An Essential Role for Cortactin in the Modulation of the Potassium Channel Kv1–2, Proc. Natl. Acad. Sci. USA, 2007, vol. 104, pp. 17412–17417.PubMedCrossRefGoogle Scholar
  50. Wu, H. and Parsons, J.T., Cortactin, an 80/85-Kilodalton Pp60src Substrate, Is a Filamentous Actin-Binding Protein Enriched in the Cell Cortex, J. Cell Biol., 1993, vol. 120, pp. 1417–1426.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • D. V. Ilatovskaya
    • 1
    • 2
  • T. S. Pavlov
    • 2
  • Yu. A. Negulyaev
    • 1
    • 3
  • A. Staruschenko
    • 2
  1. 1.Institute of Cytology RASSt. PetersburgRussia
  2. 2.Medical College of WisconsinMilwaukeeUSA
  3. 3.Faculty of Medical Physics and BioengineeringSt. Petersburg State Polytechnical UniversitySt. PetersburgRussia

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