Extracellular ATP (Ex-ATP) plays a central role in many cellular processes like Ca2+ homeostasis, cell proliferation and apoptosis. Ex-ATP binds to the plasma membrane- localized P2 receptors resulting in the activation of several downstream signaling pathways. There are two types of P2 receptors, metabotropic P2Y, and ionotropic P2X. P2Y receptors (P2YRs) are G protein-coupled receptors, whereas P2X receptors (P2XRs) are ligand-gated ion channels. P2XRs are expressed ubiquitously in many organs and control the vital functions of the central nervous system, vascular system, and immune cells. P2X7 receptor (P2X7R) is a homotrimeric trans-membrane channel with three extracellular ATP-binding pockets located at the subunit-subunit interfaces. P2X7R is involved in several cellular processes like activation of NLRP3, and kinases like MAP kinase and Src Kinase. Many cells release ATP in different physiological and pathophysiological conditions. ATP-release is generally mediated by vesicular secretion and through different ion channels. One noteworthy ATP- release channel is the Pannexin 1 (Panx1). Panx1 and P2X7R work together to control several inflammatory and cell death processes. Panx1 is a homo-heptameric channel with four trans-membrane domains, two extracellular loops, cytoplasmic N- and C- termini (CT). The CT of each subunit plugs the central pore keeping the channel closed in basal conditions. Withdrawal of CT from the pore under stimulating conditions is followed by the opening of non-selective Panx1 channel. Improper functioning of Panx1 has been linked to diseases like epilepsy, Alzheimer's disease and cancer.

A disease-associated mutation, Arg-217-His (R217H) in the 3rd trans-membrane domain of Panx1 is known to attenuate channel functions through an unknown
mechanism. Since the CT gates the channel, we hypothesized that R217 interacts with the CT, and this interaction is required for optimum channel activities. Based on this hypothesis, our first objective was set to understand the involvement of CT in determining the properties of the mutant channel. R217H mutation reduced the currents in the full-length channel but did not affect CT- truncated Panx1-Δ386 (last 40 amino acids, deleted from the CT). Compared to the wild-type, Panx1-R217H expressing cells showed lesser cell death when activated through P2X7R. However, cell death in Panx1-R217H-Δ386 and Panx1-Δ386 expressing cells were similar. The mutation had no effect unless the channel has an intact CT. Based on our results, we propose that R217H mutation perturbs the conformational flexibility of CT, leading to channel dysfunction.

ATP, released through Panx1, binds to P2X7R and triggers various signaling pathways. Panx1 and P2X7R have been shown to interact physically. Extracellular Ca2+ enters the cell through activated P2X7R in a controlled fashion. Dysregulated Ca2+ influx is often associated with several diseases. Our second objective was to investigate the effect of Panx1 on P2X7R-mediated Ca2+ influx. Panx1 attenuated P2X7R-mediated cytosolic Ca2+ ([Ca2+]c) rise in CHO-K1 and HEK-293 cells. In the same line, [Ca2+]c rise was higher in Panx1 knockdown astrocytes. The inhibitory effect was unaffected in the presence of Panx1 blocker carbenoxolone, suggesting that ion conduction through Panx1 is not required for inhibiting P2X7R. By sequentially deleting amino acids from the Panx1-CT, we found that the region between 350th and 386th amino acid residues is crucial for inhibiting P2X7R. Like full-length Panx1, the CT (350th to 426th amino acids) alone attenuated the [Ca2+]c rise. Based on these observations, we conclude that the region between 350th and 426th amino acids is the regulatory element that attenuates P2X7R.

Unlike other P2XRs, P2X7R exhibits gradual increase of channel conductance upon prolonged application of the agonist. Increased channel conductance is followed by cell death. Our third objective was to explore the protective role of Panx1-CT against P2X7R-mediated cell death. As reported by many other groups, we also found that persistent activation of P2X7R with ATP gradually increases the channel conductance. Interestingly, in the presence of Panx1-CT, P2X7R generated lesser currents at all ATP stimulations. However, P2X7R, when expressed with full-length Panx1, generated lesser currents at initial stimulations but exhibited larger currents at later stimulations. This demonstrates that Panx1 has an inhibitory effect at initial ATP stimulations but turns into an enhancer if the activation persists for a longer time. In the same line, P2X7R-mediated cell death, mitochondrial depolarization, mitochondrial and cytoplasmic ROS (reactive oxygen species) generation, and Caspase 3 activation were significantly increased by full-length Panx1 but reduced by CT alone.

Our findings suggest a dual role of Panx1. It promotes both cell survival and death, depending on the cellular context. It activates P2X7R by releasing ATP and, at the same time, plays a protective role by keeping P2X7R under control. Prolonged activation of P2X7R, which is an apoptotic stimulus, cleaves the CT of Panx1, making the latter a facilitator of cell death. Also, the removal of CT, a physiological brake, makes the P2X7R fully active, further contributing to the cell death process. As Panx1-P2X7R is directly involved in many inflammatory conditions, our findings will be useful in designing novel drugs for treating several diseases.



Publications:
[1] Purohit, R., and Bera, A. K. (2021) Pannexin 1 plays a pro-survival role by attenuating P2X7 receptor-mediated Ca2+ influx. Cell calcium. vol.99 102458. doi:10.1016/j.ceca.2021.102458
[2] Purohit, R., and Bera, A. K. (2021) Mutational effects of Pannexin 1 R217H depend on the carboxyl-terminus. Biochemical and biophysical research communications. vol. 548 143-147.doi:10.1016/j.bbrc.2021.02.060