Submitted on February 23, 2009
Revised on May 21, 2009
Accepted on May 25, 2009
EXTRACELLULAR ATP DISSOCIATES NONMUSCLE MYOSIN FROM THE P2X7 COMPLEX: THIS DISSOCIATION REGULATES P2X7 PORE FORMATION
Ben J Gu1, Catherine Rathsam1, Leanne Stokes1, Andrew B McGeachie1, and James S. Wiley2*
1 The University of Sydney
2 Nepean Hospital
* To whom correspondence should be addressed. E-mail: wileyj{at}medicine.usyd.edu.au.
The P2X7 receptor is a ligand-gated cation channel which is highly expressed on monocyte-macrophages and which mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7 channel and massive K+ efflux follows initial channel opening but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7 were isolated by using anti-P2X7 monoclonal antibody coated Dynabeads from both interferon-gamma plus LPS stimulated monocytic THP-1 cells and P2X7 transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va were found to associate with P2X7 in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7 receptor by ATP caused dissociation of P2X7 from nonmuscle myosin in both cell types. The interaction of P2X7 and NMMHC-IIA molecules was confirmed by fluorescent life time measurements (FLIM) and fluorescent resonance of energy transfer (FRET) based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by siRNA or shRNA led to a significant increase of P2X7 pore function without any increase in surface expression or ion channel function of P2X7 receptors. S-(-)-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7 and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7 channel to a pore.
