An essential role of PI(4,5)P2 for maintaining the activity of the transient receptor potential canonical (TRPC)4β

  • Hana Kim
  • Jae-Pyo Jeon
  • Chansik Hong
  • Jinsung Kim
  • Jongyoun Myeong
  • Ju-Hong Jeon
  • Insuk SoEmail author
Ion Channels, Receptors and Transporters


The transient receptor potential canonical 4 (TRPC4) channel is a Ca2+-permeable nonselective cation channel in mammalian cells and mediates a number of cellular functions. Many studies show that TRPC channels are activated by stimulation of Gαq-phospholipase C (PLC)-coupled receptors. However, our previous study showed that the TRPC4 current was inhibited by co-expression of a constitutively active form of Gαq (Gαq Q209L). A shortage of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in Gαq Q209L may be responsible for reduced TRPC4 activity. Here, we tested this hypothesis by using a rapamycin-inducible system that regulates PI(4,5)P2 acutely and specifically. Our results showed that the TRPC4β current was reduced by inducible Gαq Q209L, but not by the mutants with impaired binding ability to PLCβ. Depletion of PI(4,5)P2 by inducing the inositol polyphosphate 5-phosphatase to HEK293 cells that express TRPC4β led to an irreversible inhibition of TRPC4β currents. In contrast, inducing phosphatidylinositol 4-phosphate 5-kinase or intracellular PI(4,5)P2 application did not activate the TRPC4β current. Finally, we revealed that PI(4,5)P2 is important in delaying the desensitization of TRPC4β. Taken together, we suggest that PI(4,5)P2 is not the activator of TRPC4β activation, but it is still necessary for regulating TRPC4β activation.


TRPC4 PIP2 GPCR Rapamycin-inducible system 



Transient receptor potential canonical


Phosphoinositide phosphatidylinositol 4,5-bisphosphate


Inducible inositol polyphosphate 5-phosphatase


Guanosine 5′-O-[gamma-thio]triphosphate


Phosphatidylinositol 4-phosphate 5-kinase


Phospholipase C



We thank Dr. Won Do Heo for the CFP-FKBP-PIP5K, CFP-FKBP-Inp54p, and Lyn-FRB constructs and Dr. Carsten Schultz for mRFP-FKBP-(GKK)3-Gαq Q209L, mRFP-FKBP-(GKK)3-Gαq Q209L/H218A, and mRFP-FKBP-(GKK)3-Gαq Q209L/L254A. We acknowledge the roles of the Biomedical Imaging Center at the Seoul National University College of Medicine, Seoul, Korea. This study was supported by grant from the National Research Foundation of Korea funded by the Korea government (MEST) (2012R1A2A1A01003073).

Supplementary material

424_2013_1236_MOESM1_ESM.pptx (268 kb)
Supplementary Figure 1 We were using fluorescent microscope olympus IX70 with 1.35 N. A and 60× oil lense. HEK293 cell is cultured in confocal dish. CFP images were obtained CFP mode (excitation : 435/20 nm LED(Cool LED) ,dichroic mirror : CFP beamsplitter(Chroma), emission filter: ET470/24 nm(Chroma). RFP images were obtained RFP mode (excitation : 535/30 nm LED(cool LED), dichroic mirror : RFP beamsplitter(chroma), emission filter : ET605/70 nm(chroma). Alll imaged captured on CCD Camera (14-bit(10 MHz standard)), DR-328G-C01-SIL : Clara, ANDOR technology, USA), with exposure time of 100 ms with 2 × 2binning(645 × 519 pixels). Scale bar is 10 μm. Image processing was performed using MEtaMorph 7.6 software(molecular devices, Japan). The interval is 30 s and 10minites recording. We treated rapamycin after first frame. (PPTX 268 kb)


  1. 1.
    Cho H, Lee D, Lee SH, Ho W (2005) Receptor-induced depletion of phosphatidylinositol 4,5-bisphosphate inhibits inwardly rectifying K+ channels in a receptor-specific manner. PNAS 102:4643–4648PubMedCrossRefGoogle Scholar
  2. 2.
    Inoue T, Heo WD, Grimley JS, Wandless TJ, Meyer T (2005) An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat Methods 2:415–418PubMedCrossRefGoogle Scholar
  3. 3.
    Jeon JP, Hong C, Park EJ, Jeon JH, Cho NH, Kim IG, Choe H, Muallem S, Kim HJ, So I (2012) Selective Gαi subunits as novel direct activators of TRPC4 and TRPC5 channels. J Biol Chem 287:17029–17039PubMedCrossRefGoogle Scholar
  4. 4.
    Jeon JP, Lee KP, Park EJ, Sung TS, Kim BJ, Jeon JH, So I (2008) The specific activation of TRPC4 by Gi protein subtype. Biochem Biophys Res Commun 377:538–543PubMedCrossRefGoogle Scholar
  5. 5.
    Kim MT, Kim BJ, Lee JH, Kwon SC, Yeon DS, Yang DK, So I, Kim KW (2006) Involvement of calmodulin and myosin light chain kinase in the activation of mTRPC5 expressed in HEK cells. Am J Physiol Cell Physiol 290:1031–1040CrossRefGoogle Scholar
  6. 6.
    Kim MJ, Jeon JP, Kim HJ, Kim BJ, Lee YM, Choe H, Jeon JH, Kim SJ, So I (2008) Molecular determinant of sensing extracellular pH in classical transient receptor potential channel 5. Biochem Biophys Res Commun 365:239–245PubMedCrossRefGoogle Scholar
  7. 7.
    Kim BJ, Kim MT, Jeon JH, Kim SJ, So I (2008) Involvement of phosphatidylinositol 4,5-bisphosphate in the desensitization of canonical transient receptor potential 5. Biol Pharm Bull 31:1733–1738PubMedCrossRefGoogle Scholar
  8. 8.
    Kwon Y, Hofmann T, Montell C (2007) Integration of phosphoinositide- and calmodulin-mediated regulation of TRPC6. Mol Cell 25:491–503PubMedCrossRefGoogle Scholar
  9. 9.
    Lee KP, Jun JY, Chang IY, Suh SH, So I, Kim KW (2005) TRPC4 is an essential component of the nonselective cation channel activated by muscarinic stimulation in mouse visceral smooth muscle cells. Mol Cell 20:435–441CrossRefGoogle Scholar
  10. 10.
    Loijens JC, Boronenkov IV, Parker GJ, Anderson RA (1996) The phosphatidylinositol 4-phosphate 5-kinase family. Adv Enzym Regul 36:115–140CrossRefGoogle Scholar
  11. 11.
    Majerus PW (1996) Inositols do it all. Genes Dev 10:1051–1053PubMedCrossRefGoogle Scholar
  12. 12.
    McLaughlin S, Wang J, Gambhir A, Murray D (2002) PIP2 and proteins: interactions, organization, and information flow. Annu Rev Biophys Biomol Struct 31:151–175PubMedCrossRefGoogle Scholar
  13. 13.
    Mitchell CA, Brown S, Campbell JK, Munday AD, Speed CJ (1996) Regulation of second messengers by the inositol polyphosphate 5-phosphatases. Biochem Soc Trans 24:994–1000PubMedGoogle Scholar
  14. 14.
    Montell C (2001) Physiology, phylogeny, and functions of the TRP superfamily of cation channels. Sci STKE 90:re1Google Scholar
  15. 15.
    Otsuguro K, Tang J, Tang Y, Xiao R, Freichel M, Tsvilovskyy V, Ito S, Flockerzi V, Zhu MX, Zholos AV (2008) Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate. J Biol Chem 283:10026–10036PubMedCrossRefGoogle Scholar
  16. 16.
    Putyrski M, Schultz C (2011) Switching heterotrimeric G protein subunits with a chemical dimerizer. Chem Biol 18:1126–1133PubMedCrossRefGoogle Scholar
  17. 17.
    Qin F (2007) Regulation of TRP ion channels by phosphatidylinositol-4,5-bisphosphate. HEP 179:509–525Google Scholar
  18. 18.
    Raghu P, Usher K, Jonas S, Chyb S, Polyanovsky A, Hardie RC (2000) Constitutive activity of the light-sensitive channels TRP and TRPL in the Drosophila diacylglycerol kinase mutant, rdgA. Neuron 26:169–179PubMedCrossRefGoogle Scholar
  19. 19.
    Rohacs T (2007) Regulation of TRP channels by PIP2. Pflugers Arch 453:753–762PubMedCrossRefGoogle Scholar
  20. 20.
    Schaefer M, Plant T, Obukhov A, Hofmann T, Gudermann T, Schultz G (2000) Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5. J Biol Chem 275:17517–17526PubMedCrossRefGoogle Scholar
  21. 21.
    Schaefer M, Plant DT, Stresow N, Albrecht N, Schultz G (2000) Functional differences between TRPC4 splice variants. J Biol Chem 277:3752–3759CrossRefGoogle Scholar
  22. 22.
    So I, Kim KW (2003) Nonselective cation channels activated by the stimulation of muscarinic receptors in mammalian gastric smooth muscle. J Smooth Muscle Res 39:231–247PubMedCrossRefGoogle Scholar
  23. 23.
    Spencer DM, Wandless TJ, Schreiber SL, Crabtree GR (1993) Controlling signal transduction with synthetic ligands. Science 262:1019–1024PubMedCrossRefGoogle Scholar
  24. 24.
    Suh BC, Hille B (2008) PIP2 is a necessary cofactor for ion channel function: how and why? Annu Rev Biophys 37:175–195PubMedCrossRefGoogle Scholar
  25. 25.
    Suh BC, Inoue T, Meyer T, Hille B (2006) Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science 314:1454–1457PubMedCrossRefGoogle Scholar
  26. 26.
    Trebak M, Lemonnier L, DeHaven IW, Wedel JB, Bird SG, Putney WJ (2009) Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels. Pflugers Arch 457:757–769PubMedCrossRefGoogle Scholar
  27. 27.
    Tseng PH, Lin HP, Hu H, Wang C, Zhu MX, Chen CS (2004) The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system. Biochemistry 43:11701–11708PubMedCrossRefGoogle Scholar
  28. 28.
    Tsvilovskyy VV, Zholos AV, Aberle T, Philipp SE, Dietrich A, Zhu MX, Birnbaumer L, Freichel M, Flockerzi V (2009) Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo. Gastroenterology 137:1415–1424PubMedCrossRefGoogle Scholar
  29. 29.
    Venkatachalam K, Zheng F, Gill DL (2003) Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 278:29031–29040PubMedCrossRefGoogle Scholar
  30. 30.
    Whorton RM, MacKinnon R (2011) Crystal structure of the mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium. Cell 147:199–208PubMedCrossRefGoogle Scholar
  31. 31.
    Zeng F, McHugh D, Beech D (2006) Inhibition of human TRPC5 activity by PIP2. FASEB J 20:A329–A330Google Scholar
  32. 32.
    Zhu MH, Chae M, Kim HJ, Lee YM, Kim MJ, Jin NG, Yang DK, So I, Kim KW (2005) Desensitization of canonical transient receptor potential channel 5 by protein kinase C. Am J Physiol Cell Physiol 289:C591–C600PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Hana Kim
    • 1
  • Jae-Pyo Jeon
    • 1
  • Chansik Hong
    • 1
  • Jinsung Kim
    • 1
  • Jongyoun Myeong
    • 1
  • Ju-Hong Jeon
    • 1
  • Insuk So
    • 1
    Email author
  1. 1.Department of PhysiologySeoul National University College of MedicineSeoulRepublic of Korea

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