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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., (Copyright © 2019 The Author(s).)

P2X7 receptor has gained an increasing importance as a drug target. One important response to P2X7 receptor stimulation is the uptake of large molecular weight tracers into cells. However, mechanism for this response is not understood clearly, but it is generally believed that a nonselective large pore protein forms this P2X7 receptor-activated permeability pathway. We examined human embryonic kidney (HEK) 293 cells transfected with rat P2X7 receptors (HEK-rP2X7) and a macrophage derived cell line, RAW 264.7, that expresses an endogenous P2X7 receptor. We used confocal microscopy to investigate uptake of different types of dyes into these cells after ATP application. Stimulation of P2X7 receptors in HEK-rP2X7 cells activated two different dye uptake pathways. The first was permeable to the cationic fluorescent dyes YO-PRO-1 and TO-TO-1 but not to the anionic dyes lucifer yellow and calcein and did not require intracellular Ca2+ concentration ([Ca2+](i)) increase to be activated. The second pathway permeated only lucifer yellow and was completely dependent on [Ca2+](i) for activation. In RAW 264.7 cells, P2X7 receptor stimulation activated uptake of ethidium, YO-PRO-1, TO-TO-1, lucifer yellow, and calcein. Again, two different permeation pathways were discerned in RAW 264.7 cells: one permeated only ethidium and the other one, only lucifer yellow. We did observed no clear [Ca2+](i) dependence for these permeation pathways. Our results demonstrate that instead of a single nonselective pore, P2X7 receptor seems to activate at least two permeation pathways, one for cationic and one for anionic dyes with different activation properties.

Organic cation transporters play a critical role in the elimination of therapeutic compounds in the liver and the kidney. We used computational quantitative structure activity approaches to predict molecular features that influence interaction with the human ortholog of the organic cation transporter (hOCT1). [(3)H]tetraethylammonium uptake in HeLa cells stably expressing hOCT1 was inhibited to varying extents by a diverse set of 30 molecules. A subset of 22 of these was used to produce, using Catalyst, a pharmacophore that consisted of three hydrophobic features and a positive ionizable feature. The correlation coefficient of observed versus predicted IC(50) was 0.86 for this training set, which was superior to calculated logP alone (r = 0.73) as a predictor of hOCT1 inhibition. A descriptor-based quantitative structure-activity relationship study using Cerius(2) resulted in an equation relating five molecular descriptors to log IC(50) with a correlation coefficient of 0.95. Furthermore, a group of phenylpyridinium and quinolinium compounds were used to investigate the spatial limitations of the hOCT1 binding site. The affinity for hOCT was higher for 4-phenylpyridiniums > 3-phenylpyridiniums > quinolinium, indicating that substrate affinity was influenced by the distribution of hydrophobic mass. In addition, supraplanar hydrophobic mass was found to increase the affinity for binding hOCT1. These results indicate how a combination of computational and in vitro approaches may yield insight into the binding affinity of transporters and may be applicable to predicting these properties for new therapeutics.