Electrophysiological characteristics of Panx1 channel and its purinergic regulation by P2X7 receptor

Abstract

Background: Extracellular ATP is a key mediator of evolutionary conserved purinergic signaling, which regulates many biological effects (tissue homeostasis, immunity, inflammation, healing etc.) and is involved in the pathogenesis of diseases of the immune, nervous, respiratory, urinary and digestive systems, skeletal muscles etc. Important source of ATP in extracellular space is the active extracellular ATP release, conducted by Panx1, a mechanosensitive non-selective ion channel, expressed in various mammalian tissues (macrophages, neurons, glial, muscular, endothelial, renal cells etc.). In turn, ATP stimulation via purinergic receptors (for example, P2X7) activates Panx1 channel. While purinergic signaling and Panx1 are considered relevant pathogenic targets for potential treatment, and some Panx1 inhibitors were already clinically tested, intrinsic properties of Panx1 channel were not well characterized. Here, we estimated electrophysiology parameters of Panx1 protein and provided its I-V Characteristic Curve, and showed that purinergic stimulation of P2X7 receptor increases Panx1 current in vitro. Methods: Activity of Panx-1 channel was recorded using a cell-attached patch clamp in CHO cells, transfected with PANX1 cDNA. To confirm Panx1 channel activity, we used blocking with 75 μM probenecid by adding the drug in bath buffer flow. For drawing I-V curve, Panx1 currents were measured and averaged (mean±SE) at different voltages in 85 experiments. This cellular model mimics cells of different systems which are abnormally overexpress the Panx1 in various pathologic conditions. We also tested if Panx1 channel stimulated via purinergic P2X7 receptor with 100μM αβ-MeATP in the CHO cells, co-transfected with plasmids, encoding PANX1 and P2X7 proteins, by analyzing the channel’s NPO dynamics. Statistical significance of experimental effects on NPO was estimated with non-parametric paired Wilcoxon Signed Ranks test with using the Origin software (Northampton, MA). Results: Probenecid significantly inhibits an activity of the channels, overexpressed in PANX1-transfected CHO cells. I-V Characteristic Curve of Panx1 channel in the same cells was obtained. NPO decreased from 1.32±0.16 to 0.52±0.09 (n=28, p<<0.0001). The inhibitory effect of the drug was reversible; after washout with bath buffer flow, NPO reverted to 1.21±0.12. Treatment of CHO cells, co-transfected with PANX1 and P2X7 plasmids, with αβ-MeATP significantly stimulated an activity of Panx1 channels; NPO increased from 0.70±0.26 to 0.97±0.23, n=9, p=0.0012. The effect was probenecid-dependent (NPO=0.40±0.12, p=0.0039). Conclusion: Purinergic stimulation of cells, co-expressing Panx1 and P2X7 proteins, increases probenecid-dependent Panx1 current. These results support that Panx1 channel is targetable for pharmacological interventions in diseases with pathogenic involvement of purinergic signaling. Support: ASN Carl W. Gottschalk award, DK123266. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.