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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

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Close spatio-association of the transient receptor potential canonical 4 (TRPC4) channel with Gαi in TRPC4 activation

JongYun Myeong1, Misun Kwak1, Jae-Pyo Jeon2

  • 1Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea; and.

American Journal of Physiology. Cell Physiology
|March 20, 2015
PubMed

Insights

G protein subunit Gαi2 directly binds to and activates TRPC4 channels, a type of calcium-permeable cation channel, leading to increased calcium influx into cells.

Area of Science:

  • Molecular biology
  • Cell physiology
  • Ion channel function

Background:

  • Transient Receptor Potential Cation (TRPC) channels, including TRPC4, are activated by various stimuli, notably G protein-coupled receptors (GPCRs).
  • While Gq/phospholipase C pathways are implicated in TRPC4 activation, the precise molecular mechanisms involving other G protein subunits remain largely unknown.

Purpose of the Study:

  • To elucidate the molecular mechanisms by which G protein alpha subunits regulate TRPC4 channel activity.
  • To investigate the direct interaction between Gαi2 and TRPC4 channels.

Main Methods:

  • Utilized whole-cell patch clamp electrophysiology and Förster Resonance Energy Transfer (FRET) microscopy.
  • Employing fluorescently tagged TRPC4 (CFP) and Gαi2 (YFP) constructs in HEK293 cells.
  • Measured intracellular calcium levels using the Yellow Cameleon 6.1 (YC6.1) calcium indicator.

Main Results:

  • Demonstrated direct binding between Gαi2 and TRPC4 channels, with higher FRET efficiency observed with a constitutively active Gαi2 mutant compared to wild-type.
  • Showed increased FRET efficiency between TRPC4 and Gαi2 upon stimulation of M2 muscarinic receptors with carbachol.
  • Confirmed direct interaction of TRPC4 with Gαi2, but not Gαq, specifically when the channel is open.
  • Observed prolonged Ca(2+) influx in cells co-expressing TRPC4, Gαi2, and M2 receptors upon carbachol stimulation.

Conclusions:

  • Gαi2 directly binds to and activates TRPC4 channels.
  • This direct interaction facilitates calcium entry through TRPC4 channels, suggesting a novel regulatory pathway for TRPC4 function.
  • These findings expand our understanding of G protein regulation of ion channel activity.
Keywords:
FRETG proteinTRPC4calcium

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