A Molecular-Level Investigation of the Effect of Sodium Ions on the Binding of Opioid Ligands to their Receptors

Abstract

The ability of sodium ions to increase the binding of antagonists and decrease the binding of agonists was first suggested for opioid receptors (ORs) in the '70s, and subsequently extended to other G protein-coupled receptors (GPCRs). These early conclusions derived from saturation analyses of ligand binding, using brain tissue homogenates with or without sodium. Further studies in tissues suggested differential regulation by sodium ions, with kappa sites being less sensitive to sodium than mu- and delta-opioid receptor sites. Our latest experiments in stable transfected cell lines demonstrate that a) agonist binding to mu, delta and kappa1 receptors is equally sensitive to sodium ions, and b) sodium ions tend to stabilize the antagonist conformation of the receptor. To provide a molecular-level understanding of these findings, we carried out microsecond-scale, all-atom standard molecular dynamics simulations of recent high-resolution crystal structures of ORs in an explicit 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-water environment at high sodium concentration. In spite of significant differences in sodium ion density around unique negatively charged residues in the extracellular region of kappa receptors, these simulations reveal similar rapid permeation of a sodium ion from the receptor extracellular sides, and its stable binding to the highly conserved residue D2.50. Notably, very high-resolution, crystal structures of GPCRs have recently provided direct evidence for sodium binding to an equivalent site involving residue D2.50. Although a second sodium ion is found to bind quite stably to a unique glutamic acid in the extracellular portion of transmembrane helix 6 of the kappa receptor (a lysine in mu and a tryptophan in delta), principal component analyses of our simulations suggest a similarly stabilized conformation of ORs by sodium.