Rotavirus toxin NSP4 induces diarrhea by activation of TMEM16A and inhibition of Na+ absorption

  • Jiraporn Ousingsawat
  • Myriam Mirza
  • Yuemin Tian
  • Eleni Roussa
  • Rainer Schreiber
  • David I. Cook
  • Karl KunzelmannEmail author
Transport Physiology


Rotavirus infection is the most frequent cause for severe diarrhea in infants, killing more than 600,000 every year. The nonstructural protein NSP4 acts as a rotavirus enterotoxin, inducing secretory diarrhea without any structural organ damage. Electrolyte transport was assessed in the colonic epithelium from pups and adult mice using Ussing chamber recordings. Western blots and immunocytochemistry was performed in intestinal tissues from wild-type and TMEM16A knockout mice. Ion channel currents were recorded using patch clamp techniques. We show that the synthetic NSP4114–135 peptide uses multiple pro-secretory pathways to induce diarrhea, by activating the recently identified Ca2+-activated Cl channel TMEM16A, and by inhibiting Na+ absorption by the epithelial Na+ channel ENaC and the Na+/glucose cotransporter SGLT1. Activation of secretion and inhibition of Na+ absorption by NSP4114–135, respectively, could be potently suppressed by wheat germ agglutinin which probably competes with NSP4114–135 for binding to an unknown glycolipid receptor. The present paper gives a clue as to mechanisms of rotavirus-induced diarrhea and suggests wheat germ agglutinin as a simple and effective therapy.


Rotavirus NSP4 Ca2+-activated Cl channels TMEM16A ENaC Epithelial Na+ channels SGLT1 Colonic epithelium Diarrhea 



This study is supported by the Deutsche Forschungsgemeinschaft DFG SFB699 A6/A7, DFG KU 756/8-2, and TargetScreen2 (EU-FP6-2005-LH-037365). KK was a research fellow of the University of Sydney Medical Foundation. We thank Dr. Brian Harfe and Dr. Jason Rock (University of Gainesville, Florida, USA) for supplying TMEM16A null mice and anti-mouse TMEM16A antibody and Ms. Marisa Sousa for her help with the TRPC-knockout animals.

Supplementary material

424_2011_947_MOESM1_ESM.doc (51 kb)
ESM 1 (DOC 51.0 kb)


  1. 1.
    Almaca J, Tian Y, AlDehni F, Ousingsawat J, Kongsuphol P, Rock JR, Harfe BD, Schreiber R, Kunzelmann K (2009) TMEM16 proteins produce volume regulated chloride currents that are reduced in mice lacking TMEM16A. J Biol Chem 284:28571–28578PubMedCrossRefGoogle Scholar
  2. 2.
    Ball JM, Mitchell DM, Gibbons TF, Parr RD (2005) Rotavirus NSP4: a multifunctional viral enterotoxin. Viral Immunol 18:27–40PubMedCrossRefGoogle Scholar
  3. 3.
    Ball JM, Tian P, Zeng CQ, Morris AP, Estes MK (1996) Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272:101–104PubMedCrossRefGoogle Scholar
  4. 4.
    Brunet JP, Cotte-Laffitte J, Linxe C, Quero AM, Geniteau-Legendre M, Servin A (2000) Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration. J Virol 74:2323–2332PubMedCrossRefGoogle Scholar
  5. 5.
    Buttery JP, Kirkwood C (2007) Rotavirus vaccines in developed countries. Curr Opin Infect Dis 20:253–258PubMedCrossRefGoogle Scholar
  6. 6.
    Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra-Moran O, Galietta LJ (2008) TMEM16A, A Membrane Protein Associated With Calcium-Dependent Chloride Channel Activity. Science 322:590–594PubMedCrossRefGoogle Scholar
  7. 7.
    Cook DI, Young JA (2002) Towards a physiology of epithelial pathogens. Pflügers Arch 443:339–343PubMedCrossRefGoogle Scholar
  8. 8.
    de la Fuente R, Namkung W, Mills A, Verkman AS (2007) Small molecule screen identifies inhibitors of a human intestinal calcium activated chloride channel. Mol Pharmacol 73:758–768CrossRefGoogle Scholar
  9. 9.
    Dong Y, Zeng CQ, Ball JM, Estes MK, Morris AP (1997) The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1, 4, 5- trisphosphate production. Proc Natl Acad Sci USA 94:3960–3965PubMedCrossRefGoogle Scholar
  10. 10.
    Flores CA, Cid LP, Sepulveda FV (2010) Strain-dependent differences in electrogenic secretion of electrolytes across mouse colon epithelium. Exp Physiol 95:686–698PubMedCrossRefGoogle Scholar
  11. 11.
    Grant J, Mahanty S, Khadir A, MacLean JD, Kokoskin E, Yeager B, Joseph L, Diaz J, Gotuzzo E, Mainville N, Ward BJ (2001) Wheat germ supplement reduces cyst and trophozoite passage in people with giardiasis. Am J Trop Med Hyg 65:705–710PubMedGoogle Scholar
  12. 12.
    Grubb BR (1999) Ion transport across the normal and CF neonatal murine intestine. Am J Physiol 277:G167–G174PubMedGoogle Scholar
  13. 13.
    Halaihel N, Lievin V, Ball JM, Estes MK, Alvarado F, Vasseur M (2000) Direct inhibitory effect of rotavirus NSP4(114–135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane. J Virol 74:9464–9470PubMedCrossRefGoogle Scholar
  14. 14.
    Heerze LD, Chong PC, Armstrong GD (1992) Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin. J Biol Chem 267:25810–25815PubMedGoogle Scholar
  15. 15.
    Keusch GT, Jacewicz M (1977) Pathogenesis of Shigella diarrhea. VII. Evidence for a cell membrane toxin receptor involving beta1 leads to 4-linked N-acetyl-D-glucosamine oligomers. J Exp Med 146:535–546PubMedCrossRefGoogle Scholar
  16. 16.
    Kunzelmann K, Beesley AH, King NJ, Karupiah G, Young JA, Cook DI (2000) Influenza virus inhibits amiloride- sensitive Na+ channels in respiratory epithelia. Proc Natl Acad Sci USA 97:10282–10287PubMedCrossRefGoogle Scholar
  17. 17.
    Kunzelmann K, König J, Markovich D, King N, Karupiah G, Cook DI (2004) Acute effects of parainfluenza virus on epithelial electrolyte transport. J Biol Chem 279:48760–48766PubMedCrossRefGoogle Scholar
  18. 18.
    Kunzelmann K, Mall M (2002) Electrolyte transport in the colon: Mechanisms and implications for disease. Physiol Rev 82:245–289PubMedGoogle Scholar
  19. 19.
    Kunzelmann K, Meanger J, King NJ, Cook DI (2007) Inhibition of airway Na+ transport by respiratory syncytial virus. J Virol 81:3714–3720PubMedCrossRefGoogle Scholar
  20. 20.
    Kunzelmann K, Scheidt K, Scharf B, Ousingsawat J, Schreiber R, Wainwright BJ, McMorran B (2006) Pseudomonas flagellin inhibits Na+ transport in airway epithelia. FASEB J 20:545–546PubMedGoogle Scholar
  21. 21.
    Kunzelmann K, Sun D, König J (2004) Effect of dietary lectins on ion transport in epithelia. Br J Pharmacol 142:1–8CrossRefGoogle Scholar
  22. 22.
    Kunzelmann K, Sun J, Schreiber R, Konig J (2004) Effects of dietary lectins on ion transport in epithelia. Br J Pharmacol 142:1219–1226PubMedCrossRefGoogle Scholar
  23. 23.
    Lee IH, Campbell CR, Song SH, Day ML, Kumar S, Cook DI, Dinudom A (2009) The activity of the epithelial sodium channels is regulated by caveolin-1 via a Nedd4–2-dependent mechanism. J Biol Chem 284(19):12663–12669PubMedCrossRefGoogle Scholar
  24. 24.
    Lorrot M, Martin S, Vasseur M (2003) Rotavirus infection stimulates the Cl- reabsorption process across the intestinal brush-border membrane of young rabbits. J Virol 77:9305–9311PubMedCrossRefGoogle Scholar
  25. 25.
    Lorrot M, Vasseur M (2007) How do the rotavirus NSP4 and bacterial enterotoxins lead differently to diarrhea? Virol J 4:31PubMedCrossRefGoogle Scholar
  26. 26.
    Morris AP, Estes MK (2001) Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin. Am J Physiol 281:G303–G310Google Scholar
  27. 27.
    Morris AP, Scott JK, Ball JM, Zeng CQ, O'Neal WK, Estes MK (1999) NSP4 elicits age-dependent diarrhea and Ca2+ mediated I influx into intestinal crypts of CF mice. Am J Physiol 277:G431–G444PubMedGoogle Scholar
  28. 28.
    Moustafa MA (2003) Role of wheat germ agglutinin (WGA) in treatment of experimental cryptosporidiosis. J Egypt Soc Parasitol 33:443–456PubMedGoogle Scholar
  29. 29.
    Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K (2009) Loss of TMEM16A causes a defect in epithelial Ca2+ dependent chloride transport. J Biol Chem 284:28698–28703PubMedCrossRefGoogle Scholar
  30. 30.
    Parr RD, Storey SM, Mitchell DM, McIntosh AL, Zhou M, Mir KD, Ball JM (2006) The rotavirus enterotoxin NSP4 directly interacts with the caveolar structural protein caveolin-1. J Virol 80:2842–2854PubMedCrossRefGoogle Scholar
  31. 31.
    Pike LJ, Casey L (1996) Localization and turnover of phosphatidylinositol 4, 5-bisphosphate in caveolin-enriched membrane domains. J Biol Chem 271:26453–26456PubMedCrossRefGoogle Scholar
  32. 32.
    Puntheeranurak S, Schreiber R, Spitzner M, Ousingsawat J, Krishnamra N, Kunzelmann K (2007) Control of ion transport in mouse proximal and distal colon by prolactin. Cell Physiol Biochem 19:77–88PubMedCrossRefGoogle Scholar
  33. 33.
    Rock JR, Futtner CR, Harfe BD (2008) The transmembrane protein TMEM16A is required for normal development of the murine trachea. Dev Biol 321:141–149PubMedCrossRefGoogle Scholar
  34. 34.
    Sausbier M, Matos JE, Sausbier U, Beranek G, Arntz C, Neuhuber W, Ruth P, Leipziger J (2006) Distal Colonic K+ Secretion Occurs via BK Channels. J Am Soc Nephrol 17:1275–1282PubMedCrossRefGoogle Scholar
  35. 35.
    Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins JR, Kunzelmann K (2010) Expression and Function of Epithelial Anoctamins. J Biol Chem 285:7838–7845PubMedCrossRefGoogle Scholar
  36. 36.
    Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134:1019–1029PubMedCrossRefGoogle Scholar
  37. 37.
    Shaul PW, Anderson RG (1998) Role of plasmalemmal caveolae in signal transduction. Am J Physiol 275:L843–L851PubMedGoogle Scholar
  38. 38.
    Sonawane ND, Zhao D, Zegarra-Moran O, Galietta LJ, Verkman AS (2007) Lectin Conjugates as Potent, Nonabsorbable CFTR Inhibitors for Reducing Intestinal Fluid Secretion in Cholera. Gastroenterology 132:1234–1244PubMedCrossRefGoogle Scholar
  39. 39.
    Spitzner M, Ousingsawat J, Scheidt K, Kunzelmann K, Schreiber R (2007) Role of voltage gated K+ channels for proliferation of colonic cancer cells. FASEB J 21:35–44PubMedCrossRefGoogle Scholar
  40. 40.
    Tradtrantip L, Namkung W, Verkman AS (2009) Crofelemer, an antisecretory antidiarrheal proanthocyanidin oligomer extracted from croton lechleri, targets two distinct intestinal chloride channels. Mol Pharmacol 77:69–78PubMedCrossRefGoogle Scholar
  41. 41.
    Wright EM (1993) The intestinal Na+/glucose cotransporter. Annu Rev Physiol 55:575–589PubMedCrossRefGoogle Scholar
  42. 42.
    Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, Park SP, Lee J, Lee B, Kim BM, Raouf R, Shin YK, Oh U (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455:1210–1215PubMedCrossRefGoogle Scholar
  43. 43.
    Zeissig S, Bergann T, Fromm A, Bojarski C, Heller F, Guenther U, Zeitz M, Fromm M, Schulzke JD (2008) Altered ENaC expression leads to impaired sodium absorption in the noninflamed intestine in Crohn's disease. Gastroenterology 134:1436–1447PubMedCrossRefGoogle Scholar
  44. 44.
    Zhang M, Zeng CQ, Dong Y, Ball JM, Saif LJ, Morris AP, Estes MK (1998) Mutations in rotavirus nonstructural glycoprotein NSP4 are associated with altered virus virulence. J Virol 72:3666–3672PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jiraporn Ousingsawat
    • 1
  • Myriam Mirza
    • 1
  • Yuemin Tian
    • 1
  • Eleni Roussa
    • 3
  • Rainer Schreiber
    • 1
  • David I. Cook
    • 2
  • Karl Kunzelmann
    • 1
    Email author
  1. 1.Institut für PhysiologieUniversität RegensburgRegensburgGermany
  2. 2.Department of PhysiologyUniversity of SydneySydneyAustralia
  3. 3.Department of AnatomyUniversity of FreiburgFreiburgGermany

Personalised recommendations