Site-Directed Mutagenesis Reveals P2XR7 Extracellular Domain Residues That Facilitate Channel Activation by Histone Proteins

Abstract

Seven mammalian ionotropic purinergic receptors (P2XRs) have been identified (P2XR1-7). All P2XRs have a large extracellular domain containing a conserved extracellular ATP binding pocked, however, P2XRs vary widely in ATP affnity and desensitization kinetics. P2XR7 is unique in that it has the lowest ATP affnity (mM), does not desensitize to ATP, and has a large intracellular C-terminal domain. In our recent work, we demonstrated that histone proteins activate P2XR7 current. However, histone-evoked currents are not inhibited by selective inhibitors of P2XR7 ATP-evoked current. We hypothesized that histone proteins may bind to a distinct allosteric site in the P2XR7 extracellular domain. To test this, we expressed mouse and human P2X receptors (m.- and h.P2XR1-7) in heterologous cells (Xenopus oocytes) and measured inward cation currents using a two-electrode voltage clamp (TEVC). This allowed us to determine which P2XR channels are suffcient for histone-induced currents and provided a strategy to determine the P2XR7 residues involved in histone- and ATP-evoked currents. Histone proteins induced large inward cation currents in oocytes expressing m.P2XR7 and m.P2XR4, but not in those expressing m.P2XR1. Because histone proteins are highly alkaline, we aligned the extracellular domains of m.P2XR7, -4, and -1, and looked for acidic residues that were conserved in P2XR7 and -4 but absent in P2XR1. This approach identified 12 possible histone binding residues, of which three (D156N, E175K, and E240Q) were found to be silent mutations. Mutations at these sites decreased histone-evoked current but did not decrease ATP-evoked current. These results provide functional insight into a new allosteric mechanism of P2XR7 channel activation by histone proteins which may be used to guide the development of new pharmacologic and therapeutic strategies. NIH R00HL133451 and R01HL155180. This is the full abstract presented at the American Physiology Summit 2024 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.