Diclofenac and other Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are Competitive Antagonists of the human P2X3 Receptor
Laura GrohsLinhan ChengSaskia CönenBassam G. HaddadAstrid S. ObrechtIdil ToklucuLisa ErnstJannis KörnerGünther SchmalzingAngelika LampertJan‐Philipp MachtensRalf Hausmann
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Abstract The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC 50 values of 138.2 µM and 76.7 µM, respectively). A weaker inhibition of hP2X1R, hP2X4R and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) proved to inhibit hP2X3R, rP2X3R and hP2X7R (IC 50 values of 221µM, 264.1µM and ∼ 900µM, respectively), questioning its widespread use as a nonselective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of the hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP-application or increasing concentrations of the agonist α,β-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results strongly support a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains inhibits gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R and hP2X7R. Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac may contribute to the analgesic effect as well as the side effect of taste disturbances of diclofenac and represent an additional mode of action besides the well-known high potency COX inhibition.Keywords:
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Abstract Ion channels are critical modulators of electrical excitability in neurons and are targets of a wide variety of neurotoxicants, including metals and insecticides. The only way to directly examine effects of toxicants on ion channel function in an individual cell on a physiologically relevant time scale is to utilize patch‐clamp electrophysiological techniques. This unit presents the basics of utilizing whole‐cell patch‐clamp techniques to study effects of toxicants on voltage‐sensitive Ca 2+ channels in neurons grown in culture.
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The patch-clamp technique is a relatively new and powerful electrophysiological technique. Its resolving power is such that ion currents can be recorded through only one protein or ion-channel. The application of this technique in higher plant physiology, its principles and advantages are discussed in this paper. Furthermore, some actual results of single-channel recordings, whole cell recordings and ATPase currents are shown and examples are given of ion-channel conductance and selectivity calculations.
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KcsA potassium channel
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Patch clamp method developed more than 30 years ago is widely used for investigation of cellular excitability manifested as transmembrane ionic current and/or generation of action potentials. This technique could be applied to measurement of ionic currents flowing through individual (single) ion channels or through the whole assembly of ion channels expressed in the whole cell. Whole cell configuration is more common for measurement of ion currents and the only one enabling measurement of action potentials. This method allows detailed analysis of mechanisms and structural determinants of voltagedependent gating of ion channels as well as regulation of channel activity by intracellular signaling pathways and pharmacological agents.
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Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. We developed a specific approach that allowed us to register, for the first time, endogenous ion channels in endometrial MSCs (eMSCs) assembled in spheroids. Particularly, using the single-channel patch-clamp technique, we have recorded the activity of ion channels and observed their functional interplay in mechanosensitive clusters. Our experimental protocol could be applied for identification and studying of ion channels in 3D cell cultures.
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