Homology Models of μ-Opioid Receptor with Organic and Inorganic Cations at Conserved Aspartates in the Second and Third Transmembrane Domains

Metal ions affect ligand binding to G-protein-coupled receptors by as yet unknown mechanisms. In particular, Na+ increases the affinity for antagonists but decreases it for agonists. We had modeled the μ-opioid receptor (μR) based on the low-resolution structure of rhodopsin by G. F. X. Schertler, C. Villa, and R. Henderson (1993, Nature 362, 770–772) and proposed that metal ions may be directly involved in the binding of ligands and receptor activation (B. S. Zhorov and V. S. Ananthanarayanan, 1998, J. Biomol. Struct. Dyn. 15, 631–637). Developing this concept further, we present here homology models of μR using as templates the structure of rhodopsin elaborated by I. D. Pogozheva, A. L. Lomize, and H. I. Mosberg (1997, Biophys. J. 70, 1963–1985) and J. M. Baldwin, G. F. X. Schertler, and V. M. Unger (1997, J. Mol. Biol., 272, 144–164). Using the Monte Carlo minimization (MCM) method, we docked the Na+-bound forms of μR ligands: naloxone, bremazocine, and carfentanyl. The resultant low-energy complexes showed that the two positive charges in the protonated metal-bound ligands interact with the two negative charges at Asp3.32 and Asp2.50 (for notations, see J. A. Ballesteros and H. Weinstein, 1995, Methods Neurosci. 25, 366–426). MCM computation on morphine docked inside the model of μR by I. D. Pogozheva, A. L. Lomize, and H. I. Mosberg (1998, Biophys. J. 75, 612–634) yielded two binding modes with the ligandʹs ammonium group salt-bridged either to Asp3.32 (generally regarded as the ligand recognition site) or to Asp2.50. The latter is the presumed site for Na+ ion, which is known to modulate ligand binding. Assuming that in the low-dielectric transmembrane region of μR, organic and inorganic cations would compete for Asp3.32 and Asp2.50, we propose that ligand binding, as visualized in the above models, would first displace Na+ from Asp3.32. A subsequent progress of the ligand toward Asp2.50 would result in either the retention of Na+ at Asp2.50 in the case of antagonists or the displacement of Na+ from Asp2.50 in the case of agonists. The displaced Na+ would move toward the salt-bridged Asp3.49–Arg3.50 and disengage the salt bridge. This, in turn, would result in conformational changes at the cytoplasmic face of the receptor that facilitate the interaction with the G-protein.