Mechanistic insights into the allosteric modulation of opioid receptors by sodium ions

Abstract

The idea of sodium ions altering G-protein-coupled receptor (GPCR) ligand binding and signaling was first suggested for opioid receptors (ORs) in the 1970s and subsequently extended to other GPCRs. Recently published ultra-high-resolution crystal structures of GPCRs, including that of the δ-OR subtype, have started to shed light on the mechanism underlying sodium control in GPCR signaling by revealing details of the sodium binding site. Whether sodium accesses different receptor subtypes from the extra- or intracellular sides, following similar or different pathways, is still an open question. Earlier experiments in brain homogenates suggested a differential sodium regulation of ligand binding to the three major OR subtypes, in spite of their high degree of sequence similarity. Intrigued by this possibility, we explored the dynamic nature of sodium binding to δ-OR, μ-OR, and κ-OR by means of microsecond-scale, all-atom molecular dynamics (MD) simulations. Rapid sodium permeation was observed exclusively from the extracellular milieu, and following similar binding pathways in all three ligand-free OR systems, notwithstanding extra densities of sodium observed near nonconserved residues of κ-OR and δ-OR, but not in μ-OR. We speculate that these differences may be responsible for the differential increase in antagonist binding affinity of μ-OR by sodium resulting from specific ligand binding experiments in transfected cells. On the other hand, sodium reduced the level of binding of subtype-specific agonists to all OR subtypes. Additional biased and unbiased MD simulations were conducted using the δ-OR ultra-high-resolution crystal structure as a model system to provide a mechanistic explanation for this experimental observation.

Figure 1

Vertical views of the representative structures from the microsecond simulations of (A) δ-OR (PDB entry 4N6H), (B) μ-OR (PDB entry 4DKL), and (C) κ-OR (PDB entry 4DJH) in cartoon representation with negatively charged (Asp and Glu) residues shown as sticks. Generic numbering for TM Asp/Glu residues and loop labels for other Asp/Glu residues are provided in parentheses. Sodium occupancy during the microsecond simulations at 0.12 and 0.24 particle/ų contour levels is shown in transparent and solid blue, respectively.

Figure 2

Sodium entry pathways for (A) δ-OR, (B) μ-OR, and (C) κ-OR microsecond simulations. The pathways are composed by connecting sodium positions at 1 ns intervals, colored by simulation time (blue to green to red). They are plotted on representative receptor simulation structures, i.e., the structures with the lowest average heavy atom RMSD from all other structures in the most populated cluster of conformations sampled during dynamics, which are shown in cartoon representation with negatively charged residues shown as sticks. Generic numbering for TM Asp/Glu residues and loop naming for non-TM Asp/Glu residues are included in parentheses.

Figure 3

Sodium coordination at the orthosteric ligand and allosteric sodium binding sites. Sodium minimum distance to side chain oxygens of D3.32 (at the orthosteric ligand site, gray) or D2.50 (at the allosteric sodium site, black) for (A) δ-OR, (B) μ-OR, and (C) κ-OR microsecond simulations. Representative conformational states are indicated as states 1 (extracted at 40, 145, and 6 ns for δ-OR, μ-OR, and κ-OR, respectively) and 2 (extracted at 768, 974, and 900 ns for δ-OR, μ-OR, and κ-OR, respectively), below the distance plots. Protein backbones from simulations are colored silver. Ion-interacting residues, bound sodium, and water from the simulation are shown as silver sticks, a blue sphere, and red spheres, respectively. In comparison, the sodium atom from the δ-OR crystal (PDB entry 4N6H) is shown as a cyan sphere, while protein and water molecules from the crystal are colored gray. Ion-interacting residues are labeled with generic numbering. Sodium coordination is indicated by black dashed lines.

Figure 4

Representative main ion egress pathways derived from RAMD simulations. Pathways are shown as colored tubes, while charged δ-OR side chains that interact with the ion along the pathways are shown as sticks. The bound agonist SNC-80 is shown in ball-and-stick representation.