Species- and agonist-dependent differences in the deactivation-kinetics of P2X 7 receptors

Abstract

In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist. BzATP was more potent than ATP at the three species homologues and exhibited highest potency for rat P2X7 receptors suggesting that channel closure time was related to agonist potency. Furthermore, BzATP potency for the P2X7 receptor could be modified by changing extracellular ionic concentrations or by mutating the receptor and modifications which increased agonist potency also increased the time taken for channel closure. The dependence of channel closure time on agonist potency suggests it reflects agonist dissociation from the P2X7 receptor rather being an intrinsic property of the ion-channel. Consistent with this, our previous studies have shown that agonist potency increases after repeated agonist applications and in this study channel closure time at rat P2X7 receptors increased after repeated agonist applications. Overall these results suggest that the species differences in channel closure times reflect differences in agonist dissociation rates which arise as a consequence of the marked species differences in agonist potency.

Genetically dissecting P2rx7 expression within the central nervous system using conditional humanized mice

“…Here, we compared the Yo-Pro-1 uptake capacity of murine and humanized P2X7R endogenously expressed in primary cells obtained from respective mice. The detected difference in Yo-Pro-1 uptake between humanized and mouse P2X7R was comparable to previous reports [8]. To activate the pore formation via the murine P2X7R to levels comparable with the human P2X7R ~10 times higher BzATP concentrations were required.…”

“…In addition, we observed that the modulator TFP had a potentiating effect on Yo-Pro-1 uptake exclusively on the murine receptor but not on the humanized P2X7R. Species-specific effects of positive and negative modulators have repeatedly been described for P2X7R orthologs [8, 10, 57, 58]. These findings suggest that our humanized mouse model is well-suited to discriminate properties of mouse and human P2X7R orthologs in an in vivo context and thereby opens new possibilities for the screening and evaluation of new P2X7R agonists and antagonists.…”

“…We used the Yo-Pro-1 uptake assay as a well-established readout for the assessment of P2X7R sensitivity toward different agonists and antagonists as well as for the comparison of receptor orthologs from different species [8, 9, 11]. All previous studies investigating inter-species differences of P2X7R orthologs were conducted in heterologously expressing cell lines.…”

“…Despite the fact that human and murine P2X7 share about 80 % sequence homology, differences between the species have been described with regards to receptor sensitivity toward various ligands. The human P2X7R has been shown to be 10–100 times more sensitive to stimulation by the agonist 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′-triphosphate (BzATP) compared to the murine ortholog [4, 8, 9]. Moreover, it has been demonstrated that human and murine receptors show different susceptibility regarding modulation of their activity by various compounds [9–11].…”

Activation of P2X7 receptor and NLRP3 inflammasome assembly in hippocampal glial cells mediates chronic stress-induced depressive-like behaviors

“…A brain infusion cannula was bilaterally implanted in the hippocampus (AP = −3.8, ML = ±3 mm, DV = 3.5 mm, 1 μl, 0.5 μl min −1 ). Seven days after implantation, vehicle, ATP (100 nM), BzATP (10.5 nM), BBG (Brilliant Blue G, one of the safest dyes currently used, is a well-known P2X7 receptor antagonist,1 pM), or A438079 (a selective competitive antagonist for the human and rodent P2X7 receptor with good bioavailability and CNS penetration widely used in animal models of disease, 1.75 nM) was delivered into the hippocampus of free-moving rats for 21 days [39–49]. The behavioral test was conducted before the infusion on the first day and 30 min following the infusion on the 21th day.…”