Ginsenosides act as positive modulators of P2X4 receptors

Kshitija Dhuna, Matthew Felgate, Stefan Bidula, Samuel Walpole, Lucka Bibic, Brett Cromer, Jesus Angulo, Julie Sanderson, Martin Stebbing, Leanne Stokes (Lead Author)

Research output: Contribution to journalArticle

12 Citations (Scopus)
14 Downloads (Pure)

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 2 or ATP 1 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.

Original languageEnglish
Pages (from-to)210-221
Number of pages12
JournalMolecular Pharmacology
Volume95
Issue number2
Early online date13 Dec 2018
DOIs
Publication statusPublished - Jan 2019

Keywords

  • ATP receptors
  • P2X receptors
  • Nucleotides
  • Docking proteins
  • Gene editing/CRISPR
  • Drug discovery

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