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.
Footnotes
- Received July 23, 2018.
- Accepted December 10, 2018.
This work was partly supported by a strategic grant from RMIT University Health Innovations Research Institute, a RMIT University Vice Chancellors Research Fellowship, a University of East Anglia School of Pharmacy start-up fund, and a Biotechnology and Biological Sciences Research Council project grant awarded to L.S. [BB/N018427/1]. K.D. was supported by a RMIT University PhD scholarship (2014–2018). J.A. and S.W. are supported by a Biotechnology and Biological Sciences Research Council project grant [BB/P010660/1] and a Biotechnology and Biological Sciences Research Council–funded NRPDTP studentship [1654460], respectively.
Data sharing
Requests for raw experimental data from this study should be directed to the corresponding author.
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- Copyright © 2019 The Author(s).
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