Date of Award

4-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Daniel Mohr Collier, Ph.D.

Committee

Julio F. Cordero-Morales, PhD; Marie Dennis M Leo, PhD; Frank Park, PhD; Udai Singh, PhD

Keywords

P2XR; P2X1; P2X2; P2X3; P2X4; P2X5; P2X7; ATP; BzATP; Histone; LL-37; docking

Abstract

P2X receptors (P2XRs) are a family of ionotropic purinergic receptors composed of seven subtypes (P2XR1–P2XR7). These trimeric, ligand-gated, non-selective cation channels are activated by adenosine triphosphate (ATP). While all P2X receptors respond to ATP, they exhibit significant variability in sensitivity, desensitization kinetics, and roles in cellular signaling. Among them, P2XR7 is unique due to its low ATP sensitivity, requiring approximately 1 mM ATP for half-maximal activation. This suggests that P2XR7 activation may necessitate alternative activation mechanisms or proximity to an ATP source, as observed in inflammatory microenvironments where P2XR7 plays a well-established role in inducing non-infectious inflammation. ATP, a damage-associated molecular pattern (DAMP), faces challenges in reaching the high extracellular concentrations required for P2XR7 activation due to rapid enzymatic degradation by ATPases. This raises questions about how P2XR7 achieves functional activation in such conditions. To address this gap, we investigate whether P2XR7 activation can be influenced by other extracellular molecules that accumulate in damaged microenvironments. Specifically, we focus on circulating histones and antimicrobial peptides (AMPs), such as LL-37, which are known DAMPs that increase at inflammatory sites following cell damage and innate immune cell activation. Extracellular histones have been shown to induce Ca²⁺ influx and propidium iodide (PI) uptake in endothelial cells, suggesting the involvement of a non-selective cation channel, although the exact mechanism remains unclear. Similarly, LL-37 has been implicated in mediating P2XR7-dependent Ca²⁺ influx and dye uptake in innate immune cells, but direct functional assays to confirm its interaction with P2XR7 are lacking. Our core hypothesis proposes that, in addition to ATP, P2XR7 can be activated by non-nucleotide agonists—such as circulating histone proteins and LL-37 peptides—through direct interaction with the receptor's extracellular domain. To test these hypotheses, we expressed the receptors of interest in Xenopus oocytes and employed two-electrode voltage clamp techniques to measure evoked currents in response to ATP, BzATP, histones, and LL-37. Our findings identify histones and LL-37 as novel agonists for P2XR7. We demonstrate that histones also activate P2XR1 and P2XR5 channels. Through combining current recording and site-directed mutagenesis, we identified five potential histone-binding sites on P2XR7, whose mutations significantly reduced histone sensitivity while preserving ATP responsiveness. Notably, P2XR7 activation by non-ATP agonists was not inhibited by highly selective allosteric inhibitors, highlighting a unique mechanism of activation. These findings may elucidate the previously unknown molecular mechanism behind histone-induced Ca²⁺ influx and PI uptake in endothelial cells, providing evidence for a direct interaction between LL-37 and P2XR7. Understanding how P2XR7 is activated by non-nucleotide agonists, such as histones and AMPs, opens new avenues for developing selective antagonists to regulate its function in various pathological conditions. Currently, no selective P2XR7 antagonists are available on the market. Additionally, the existing selective inhibitors only block ATP-mediated activation of the channel, without affecting the activation induced by non-ATP agonists, as demonstrated here.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0001-8035-1959

DOI

10.21007/aetd.cghs.2025.0017

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