Calcium-dependent block of P2X7 receptor channel function is allosteric

Abstract

Among purinergic P2X receptor (P2XR) channels, the P2X7R exhibits the most complex gating kinetics; the binding of orthosteric agonists at the ectodomain induces a conformational change in the receptor complex that favors a gating transition from closed to open and dilated states. Bath Ca2+ affects P2X7R gating through a still uncharacterized mechanism: it could act by reducing the adenosine triphosphate4− (ATP4−) concentration (a form proposed to be the P2X7R orthosteric agonist), as an allosteric modulator, and/or by directly altering the selectivity of pore to cations. In this study, we combined biophysical and mathematical approaches to clarify the role of calcium in P2X7R gating. In naive receptors, bath calcium affected the activation permeability dynamics indirectly by decreasing the potency of orthosteric agonists in a concentration-dependent manner and independently of the concentrations of the free acid form of agonists and status of pannexin-1 (Panx1) channels. Bath calcium also facilitated the rates of receptor deactivation in a concentration-dependent manner but did not affect a progressive delay in receptor deactivation caused by repetitive agonist application. The effects of calcium on the kinetics of receptor deactivation were rapid and reversible. A438079, a potent orthosteric competitive antagonist, protected the rebinding effect of 2’(3′)-O-4-benzoylbenzoyl)ATP on the kinetics of current decay during the washout period, but in the presence of A438079, calcium also increased the rate of receptor deactivation. The corresponding kinetic (Markov state) model indicated that the decrease in binding affinity leads to a decrease in current amplitudes and facilitation of receptor deactivation, both in an extracellular calcium concentration–dependent manner expressed as a Hill function. The results indicate that calcium in physiological concentrations acts as a negative allosteric modulator of P2X7R by decreasing the affinity of receptors for orthosteric ligand agonists, but not antagonists, and not by affecting the permeability dynamics directly or indirectly through Panx1 channels. We expect these results to generalize to other P2XRs.

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

“…This suggests that millimolar (or perhaps micromolar) concentrations of extracellular Ca 2+ and Mg 2+ may act as allosteric inhibitors of murine P2X7 as originally observed for rat P2X7 [46] and established by more recent studies of rat P2X7 [28,29]. The current study also demonstrates that an increase in intracellular Ca 2+ is not essential for P2X7-induced ROS formation (and pore formation) in MEL cells.…”

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

“…3, A and B). We used ATP because the cost of using millimolar concentrations of BzATP was prohibitive, and because others have shown that ATP and BzATP bind Ca 2ϩ equally well (8 . We calculated the extracellular concentration of free Ca 2ϩ in a given concentration of BzATP or ATP using MaxChelator (Fig.…”

“…The molecular physiology of the Ca 2ϩ flux is poorly understood despite outstanding recent advances in functional and structural studies (7). This is particularly true for the P2X7 receptor (P2X7R) that is unusually sensitive to allosteric block by extracellular Ca 2ϩ (8), a fact that makes characterization of the Ca 2ϩ component of the ATP-gated current particularly problematic (9,10).…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…Two hypothetical mechanisms, namely the gating model and the Pannexin-1 model, have been proposed [3] . In the gating model, pore dilation resulted from longterm ATP action, which leads to additional conformational change [118][119][120][121][122][123][124] . While in the Pannexin-1 model, the conformational changes of the P2X receptor resulted in allosterism of an auxiliary protein coupled with the P2X receptor, such as Pannexin-1, permitting molecules to enter cells through those proteins [125,126] .…”