Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7

Stefan M Bidula,Brett A Cromer,Leanne Stokes,Jesus Angulo,Samuel Walpole

doi:10.1038/s41598-019-39771-5

Abstract

P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body β-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels.

Highlights

P2X7 is an ATP-gated ion channel expressed on various immune cell populations including monocytes and macrophages[1]
We previously demonstrated that ginsenoside Rd and the major in vivo metabolite compound K (CK), act as positive allosteric modulators of human P2X7 receptors requiring the presence of agonist (ATP) for their action
The best predicted binding site for ginsenosides is located within the central vestibule of human P2X7 (hP2X7) in a large hydrophobic cleft lined by β-sheets that define the lower body wall of the trimeric channel (Fig. 1)

Summary

Introduction

P2X7 is an ATP-gated ion channel expressed on various immune cell populations including monocytes and macrophages[1]. We recently identified that the activity of P2X7 receptors could be modulated by protopanaxadiol ginsenosides such as CK, Rd, Rb1 and Rh213. These chemicals could enhance the action of the physiological agonist ATP, increasing both the sensitivity and the maximum response of P2X7 to agonist, defining the ginsenosides as positive allosteric modulators[13]. There have been few positive allosteric modulators (PAMs) identified for P2X7 including clemastine, polymyxin B, ivermectin and tenidap[14,15,16] and currently nothing is known about their molecular binding sites. Our aim in this study was to identify the putative molecular binding site for ginsenoside PAMs on the P2X7 receptor using a molecular modelling approach combined with site-directed mutagenesis. We demonstrate that mutations of two key residues, D318 and L320, abolished potentiation of P2X7 responses by the ginsenosides CK and Rd

Methods

Results

Conclusion