Pflügers Archiv - European Journal of Physiology

, Volume 467, Issue 8, pp 1723–1732 | Cite as

Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca2+-activated non-selective cation channel TRPM4

  • Oscar Cerda
  • Mónica Cáceres
  • Kang-Sik Park
  • Elías Leiva-Salcedo
  • Aníbal Romero
  • Diego Varela
  • James S. Trimmer
  • Andrés Stutzin
Ion channels, receptors and transporters


Transient receptor potential melastatin-like 4 (TRPM4) is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.


TRP channels LC-MS/MS Basolateral Phosphorylation Casein kinase 



Transient receptor potential melastatin-like 4


Phosphatidylinositide 4, 5-bisphosphate


Protein kinase C


Liquid chromatography coupled-tandem mass spectrometry



We thank Dr. Pierre Launay for providing the pcDNA4/TO-hTRPM4. We also thank Dr. David Virshup for providing the 4HA-CKIε and 4HA-CKIε (K38R) plasmids (via Addgene, plasmids 13724, and 13725, respectively). We are grateful to Dr. Jon Sack and Ms. Ashleigh Evans for the discussion and critical comments to the manuscript. We also thank to Dr. Ricardo Armisén for constructive discussions and Ms. Heidi Pérez, Mr. Nicanor Villarroel and Mr. Francisco Alfaro for technical support. Mass spectrometry was performed at the University of California Davis Proteomics Facility. FONDECYT 11121239 to O.C., National Institutes of Health Grant NS42225 to J.S.T and FONDAP 15010006 to A.S. funded this research. FONDECYT 3120041 Postdoctoral Grant supported M.C. MECESUP UCH0301 Doctoral Fellowship supported E.L.S. and Conicyt Doctoral Fellowship funded A.R.

Author’s contributions

O.C., J.S.T., and A.S. designed research. O.C., M.C., K-S.P, E.L-S., and A.R. performed research. O.C., M.C., K-S.P, E.L-S, D.V., J.S.T, and A.S. analyzed data. O.C., J.S.T., and A.S. wrote the manuscript.

Conflict of interest

The authors declare no conflict of interests.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Oscar Cerda
    • 1
    • 2
    • 3
  • Mónica Cáceres
    • 1
    • 3
  • Kang-Sik Park
    • 3
    • 4
  • Elías Leiva-Salcedo
    • 2
    • 5
  • Aníbal Romero
    • 1
  • Diego Varela
    • 2
    • 5
  • James S. Trimmer
    • 3
    • 6
  • Andrés Stutzin
    • 2
    • 5
  1. 1.Programa de Biología Celular y Molecular, Facultad de Medicina, ICBMUniversidad de ChileSantiagoChile
  2. 2.Centro de Estudios Moleculares de la Célula (CEMC), Facultad de MedicinaUniversidad de ChileSantiagoChile
  3. 3.Department of Neurobiology, Physiology and Behavior, College of Biological SciencesUniversity of CaliforniaDavisUSA
  4. 4.Department of PhysiologyKyung Hee University School of MedicineSeoulSouth Korea
  5. 5.Programa de Fisiopatología, Facultad de Medicina, ICBMUniversidad de ChileSantiagoChile
  6. 6.Department of Physiology and Membrane Biology, School of MedicineUniversity of CaliforniaDavisUSA

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