Dynamics of P2X7 receptor pore dilation:: Pharmacological and functional consequences

The biophysical and functional properties of the human P2X(7) receptor, expressed recombinantly in HEK-293 cells or natively in THP-1 pro-monocytic cells, were investigated in the context of pore dilation and externalisation of mature interleukin 1 beta (IL1 beta). In HEK-293 cells, the agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) caused concentration-dependent inward currents (EC50 59 muM) and with prolonged application this agonist caused a gradual increase in inward current culminating in a plateau. This increase in current was associated with pore dilation, determined by intracellular accumulation of YO-PRO-1. BzATP displayed increased potency at the pore-dilated form of the P2X(7) receptor (EC50 17 muM), and positive correlations between apparent receptor density and speed of pore dilation were observed. A monoclonal antibody selectively blocked current mediated by the naive receptor, while currents through pore-dilated receptors were not significantly affected, which together suggest a conformational change at the level of the receptor during the dilation event. The release of mature IL1 beta from THP-1 cells was independent of P2X(7)-mediated cell lysis, as determined by study of lactate dehydrogenase release. Moreover, using conditions designed to minimise pore dilation (using buffers containing 2 mM Ca2+ and 1 mm Mg2+), BzATP caused significant release of IL1 beta, but without concomitant YO-PRO-1 accumulation, indicating pore dilation is not required for IL1 beta release. In addition, short (4-min) incubation of THP-1 cells with BzATP (terminated by enzymatic degradation of BzATP using apyrase) resulted in significant quantities of IL1 beta release some 60 min later, suggesting commitment of cells to release IL1 beta can be triggered with only brief receptor ligation. These findings suggest that receptor expression and ligation time are critical factors for selecting multiple functional states of P2X(7). (C) 2001 Wiley-Liss, Inc.