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Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
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Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

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Negative regulation of TRPC3 channels by protein kinase C-mediated phosphorylation of serine 712

Mohamed Trebak1, Nadine Hempel, Barbara J Wedel

  • 1Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. trebak@niehs.nih.gov

Molecular Pharmacology
|November 10, 2004
PubMed

Insights

Transient receptor potential canonical 3 (TRPC3) channels are regulated by protein kinase C (PKC). Serine 712 phosphorylation by PKC provides negative feedback, controlling TRPC3 channel activity and calcium (Ca2+) entry.

Area of Science:

  • Molecular biology
  • Cell physiology
  • Ion channel function

Background:

  • Transient receptor potential canonical 3 (TRPC3) channels are nonselective cation channels activated by phospholipase C-coupled receptors.
  • Diacylglycerol (DAG) analogs like OAG activate TRPC3 independently of protein kinase C (PKC).
  • Phorbol 12-myristate 13-acetate (PMA) inhibits OAG-mediated TRPC3 activation, suggesting PKC-mediated negative feedback.

Purpose of the Study:

  • To investigate the role of PKC phosphorylation in regulating TRPC3 channel activity.
  • To identify the specific site of PKC phosphorylation responsible for negative feedback.
  • To elucidate the dual role of diacylglycerol in TRPC3 channel regulation.

Main Methods:

  • Site-directed mutagenesis to alter the phosphorylation site on TRPC3.
  • In vivo phosphorylation assays using PMA treatment.
  • Measurement of TRPC3 channel activity and calcium (Ca2+) influx in HEK293 cells.
  • Comparison of wild-type TRPC3 and S712A mutant channel function.

Main Results:

  • PMA treatment induced phosphorylation of wild-type TRPC3 channels in vivo.
  • Mutation of Serine 712 (S712A) abolished PMA-induced phosphorylation and negative feedback.
  • The S712A TRPC3 mutant exhibited significantly greater Ca2+ entry compared to wild-type TRPC3.
  • PKC-mediated phosphorylation at Serine 712 is essential for negative regulation of TRPC3.

Conclusions:

  • Diacylglycerol acts as a dual signal, activating TRPC3 channels and simultaneously triggering negative feedback via PKC phosphorylation.
  • Serine 712 is a critical site for PKC-mediated inhibition of TRPC3 channel activity.
  • Understanding this regulatory mechanism is crucial for comprehending TRPC3 channel function in cellular signaling.

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