Orthosteric (BzATP) facilitation and allosteric (Ca²⁺) inhibition of P2X7R. (A) In resting conditions (no agonist bound), the naive P2X7R is a symmetrical trimer, with three binding sites having equal and high affinity for orthosteric agonists, and is closed. The occupation of one binding site by agonist does not open the pore but leads to the loss of symmetry and an assumed reduction in the affinity of the two remaining binding sites as a result of a conformational change. When a second site is occupied, the affinity of the remaining binding site is further reduced, but the channel opens in a low conductance state. When three binding sites are occupied, however, symmetry is restored, and the binding rates revert to those of a naked receptor, allowing the pore to gradually dilate to a high conductance state and to sensitize. (B) The naive receptor in the open state generates monophasic currents with peak amplitude determined by BzATP concentration. The naive receptor at high BzATP concentration dilates and generates high amplitude biphasic currents. (C) Dependence of the peak current amplitude, measured 40 s after agonist application, on orthosteric and allosteric agonist concentrations. Gray trace, concentration dependence on BzATP of the peak current response of naive receptors in the presence of physiological bath Ca²⁺ concentration. Red trace, concentration dependence on BzATP of the peak current response of sensitized receptors, termed orthosteric facilitation, in the presence of physiological Ca²⁺ concentration. Green trace, concentration dependence on BzATP of the peak current response of naive receptors in the presence of elevated bath Ca²⁺. For details on orthosteric facilitation, see Yan et al. (2010). Experiments presented in this manuscript show that the rightward shift in the potency of agonist to activate P2X7R reflects allosteric inhibition of the receptor by this cation.