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RECEPTORS AND SIGNAL TRANSDUCTION

Extracellular ATP dissociates nonmuscle myosin from P2X₇ complex: this dissociation regulates P2X₇ pore formation

¹Department of Medicine, Nepean Clinical School, Penrith; ²Institute of Dental Research, Westmead; ³Cell Signalling Unit, Children's Medical Research Institute, Westmead, The University of Sydney, Australia

Submitted 23 February 2009 ; accepted in final form 25 May 2009

The P2X₇ receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that 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- 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 and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X₇ pore function without any increase in surface expression or ion channel function of P2X₇ receptors. S-l-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.

short hairpin RNA; blebbistatin; THP-1 cells; fluorescent resonance energy transfer; ethidium uptake; myosin IIA; myosin Va; phagocytosis