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Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells
11:47

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Published on: April 3, 2019

Extracellular ATP dissociates nonmuscle myosin from P2X(7) complex: this dissociation regulates P2X(7) pore formation

Ben J Gu1, Catherine Rathsam, Leanne Stokes

  • 1Department of Medicine, Nepean Clinical School, Penrith, NSW, Australia.

American Journal of Physiology. Cell Physiology
|June 5, 2009
PubMed

Insights

Extracellular ATP binding to the P2X(7) receptor causes nonmuscle myosin to dissociate, which is crucial for forming a larger pore. This finding reveals a new mechanism for P2X(7) receptor pore function.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • The P2X(7) receptor (P2X7R) is a key mediator of inflammation, activated by extracellular ATP.
  • P2X7R activation leads to cation channel dilation and K+ efflux, but the mechanism of secondary pore formation remains unclear.

Purpose of the Study:

  • To identify proteins associated with P2X7R.
  • To elucidate the role of these proteins in P2X7R pore formation.

Main Methods:

  • Co-immunoprecipitation using anti-P2X7R antibodies from stimulated THP-1 cells and transfected HEK-293 cells.
  • RNA interference (siRNA/shRNA) to reduce nonmuscle myosin expression.
  • Inhibition of nonmuscle myosin ATPase activity with S-l-blebbistatin.
  • Fluorescence-based assays to confirm protein interactions.

Main Results:

  • Nonmuscle myosins (NMMHC-IIA and myosin Va) were identified as P2X7R-associated proteins.
  • ATP stimulation caused the dissociation of P2X7R from nonmuscle myosins.
  • Reduced nonmuscle myosin levels significantly enhanced P2X7R pore function.
  • Inhibition of NMMHC-IIA ATPase blocked ATP-induced pore formation.

Conclusions:

  • Nonmuscle myosins act as anchoring proteins for P2X7R.
  • ATP-induced dissociation of nonmuscle myosin from P2X7R is essential for secondary pore formation.
  • This mechanism provides new insights into P2X7R-mediated inflammatory signaling.

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