Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor

Introduction: 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.Methods: 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.Results: We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 and 76.7 µM, respectively). A weaker inhibition of hP2X1R, hP2X4R, and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) inhibited hP2X3R, rP2X3R, and hP2X7R (IC50 values of 221 µM, 264.1 µM, and ∼900 µM, respectively), calling into question its use as a non-selective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of 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 suggest a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the 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.Discussion: Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac, which are rarely reached in the therapeutic range, may play a minor role in analgesia compared to the high-potency cyclooxygenase inhibition but may explain the known side effect of taste disturbances caused by diclofenac

Insights into the function of ion channels by computational electrophysiology simulations

Ion channels are of universal importance for all cell types and play key roles in cellular physiology and pathology. Increased insight into their functional mechanisms is crucial to enable drug design on this important class of membrane proteins, and to enhance our understanding of some of the fundamental features of cells. This review presents the concepts behind the recently developed simulation protocol Computational Electrophysiology (CompEL), which facilitates the atomistic simulation of ion channels in action. In addition, the review provides guidelines for its application in conjunction with the molecular dynamics software package GROMACS. We first lay out the rationale for designing CompEL as a method that models the driving force for ion permeation through channels the way it is established in cells, i.e., by electrochemical ion gradients across the membrane. This is followed by an outline of its implementation and a description of key settings and parameters helpful to users wishing to set up and conduct such simulations. In recent years, key mechanistic and biophysical insights have been obtained by employing the CompEL protocol to address a wide range of questions on ion channels and permeation. We summarize these recent findings on membrane proteins, which span a spectrum from highly ion-selective, narrow channels to wide diffusion pores. Finally we discuss the future potential of CompEL in light of its limitations and strengths. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov

Membrane Potentials Regulating GPCRs:Insights From Experiments and Molecular Dynamics Simulation

G-protein coupled receptors (GPCRs) form the largest class of membrane proteins in humans and the targets of most present drugs. Membrane potential is one of the defining characteristics of living cells. Recent work has shown that the membrane voltage, and changes thereof, modulates signal transduction and ligand binding in GPCRs. As it may allow differential signalling patterns depending on tissue, cell type, and the excitation status of excitable cells, GPCR voltage sensitivity could have important implications for their pharmacology. This review summarises recent experimental insights on GPCR voltage regulation and the role of molecular dynamics simulations in identifying the structural basis of GPCR voltage-sensing. We discuss the potential significance for drug design on GPCR targets from excitable and non-excitable cells

Structural mechanisms of voltage sensing in G protein coupled receptors

G-protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins and one-third of all drug targets in humans. A number of recent studies have reported evidence for substantial voltage regulation of GPCRs. However, the structural basis of GPCR voltage sensing has remained enigmatic. Here, we present atomistic simulations on the δ-opioid and M2 muscarinic receptors, which suggest a structural and mechanistic explanation for the observed voltage-induced functional effects. The simulations reveal that the position of an internal Na+ ion, recently detected to bind to a highly conserved aqueous pocket in receptor crystal structures, strongly responds to voltage changes. The movements give rise to gating charges in excellent agreement with previous experimental recordings. Furthermore, free energy calculations show that these rearrangements of Na+ can be induced by physiological membrane voltages. Due to its role in receptor function and signal bias, the repositioning of Na+ has important general implications for signal transduction in GPCRs