Dynamics of P2X(7) receptor pore dilation: Pharmacological and functional consequences

The biophysical and functional properties of the human P2X(7) receptor, exp ressed recombinantly in HEK-293 cells or natively in THP-1 pro-monocytic ce lls, 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 inwa rd currents (EC50 59 muM) and with prolonged application this agonist cause d a gradual increase in inward current culminating in a plateau. This incre ase in current was associated with pore dilation, determined by intracellul ar accumulation of YO-PRO-1. BzATP displayed increased potency at the pore- dilated form of the P2X(7) receptor (EC50 17 muM), and positive correlation s between apparent receptor density and speed of pore dilation were observe d. A monoclonal antibody selectively blocked current mediated by the naive receptor, while currents through pore-dilated receptors were not significan tly affected, which together suggest a conformational change at the level o f the receptor during the dilation event. The release of mature IL1 beta fr om THP-1 cells was independent of P2X(7)-mediated cell lysis, as determined by study of lactate dehydrogenase release. Moreover, using conditions desi gned to minimise pore dilation (using buffers containing 2 mM Ca2+ and 1 mm Mg2+), BzATP caused significant release of IL1 beta, but without concomita nt 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 BzA TP (terminated by enzymatic degradation of BzATP using apyrase) resulted in significant quantities of IL1 beta release some 60 min later, suggesting c ommitment of cells to release IL1 beta can be triggered with only brief rec eptor ligation. These findings suggest that receptor expression and ligatio n time are critical factors for selecting multiple functional states of P2X (7). (C) 2001 Wiley-Liss, Inc.