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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

Abstract

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.

Keywords

TRPC4 PIP2 GPCR Rapamycin-inducible system 

Abbreviations

TRPC

Transient receptor potential canonical

PI(4,5)P2

Phosphoinositide phosphatidylinositol 4,5-bisphosphate

Inp54p

Inducible inositol polyphosphate 5-phosphatase

GTPγS

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

PIP5K

Phosphatidylinositol 4-phosphate 5-kinase

PLC

Phospholipase C

Notes

Acknowledgments

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)

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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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