Ginsenosides Act As Positive Modulators of P2X4 Receptors.

Kshitija Dhuna,Matthew Felgate,Brett A Cromer,Stefan M Bidula,Samuel Walpole,Lucka Bibic,Jesus Angulo,Julie Sanderson,Leanne Stokes,Martin J Stebbing

doi:10.1124/mol.118.113696

Abstract

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP− or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.

Highlights

P2X receptors are a family of ATP-gated nonselective cation channels of which there are seven known subunits (P2X1–7) with varying expression patterns (North, 2002)
In our previous study (Helliwell et al, 2015), we demonstrated that the major ginsenoside metabolite generated in vivo, compound K (CK), in addition to protopanaxadiol ginsenosides Rd, Rb1, and Rh2, acts on P2X7 to potentiate the action of ATP
The ATP-induced YOPRO-1 dye uptake response could be increased by ivermectin (3–5 mM) and reduced by the known P2X4 antagonist 5-BDBD (20 mM) (Fig. 1)

Summary

Introduction

P2X receptors are a family of ATP-gated nonselective cation channels of which there are seven known subunits (P2X1–7) with varying expression patterns (North, 2002). Their physiological roles range from the regulation of membrane potential and intracellular calcium concentration (all P2X receptors) to the regulation of mediator secretion such as interleukin 1b (IL-1b) and brain-derived neurotrophic factor (BDNF) by P2X7 and P2X4, respectively.

Methods

Results

Conclusion