The P2X7 receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules (> 600 Da). Activation of this large pore can lead to cell lysis within 1-2 min. We asked whether pharmacological differences existed between the cationic channel and the cell permeabilizing pore by measuring whole-cell currents and uptake of a propidium dye (YO-PRO; Mw 629) in HEK293 cells stably expressing the rat P2X7 receptor, and comparing the actions of divalent cations and protons in these two assays. Currents in response to 2'-3'-(O)-(4-benzoyl benzoyl) ATP (BzATP, 30 microM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC50) of 2.9 mM, 0.5 mM, 11 microM, 0.5 microM and 0.4 microM, respectively. The inhibition was voltage independent in each case. YO-PRO uptake induced by BzATP was also inhibited with similar IC50 values. The rank order of potency of a range of divalents was Cu2+ > Cd2+ = Zn2+ > Ni2+ >> Mg2+ = Co2+ > Mn2+ > Ca2+ = Ba2+ >> Sr2+. These results suggest that these divalent cations and protons all act primarily as allosteric modulators to alter the affinity of ATP binding to the P2X7 receptor. In contrast, extracellular (but not intracellular) calmidazolium inhibited the BzATP-evoked current by up to 90% (IC50 = 15 nM) but had no effect on YO-PRO uptake. Thus, calmidazolium can block activation of the ionic channel but this does not prevent the formation of the large permeabilizing pore.