AbstractPlaying a central role in cell signaling, G-protein coupled receptors (GPCRs) form the largest superfamily of membrane proteins and approximately a third of all drug targets in humans. The 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.
However, the structural basis of GPCR voltage sensing has remained enigmatic. Here, I present atomistic simulations on the Rhodopsin GPCR family, 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 of a Na+ ion or a proton from the Na+ binding site gives rise to gating charges in excellent agreement with previous experimental recordings.
Structural studies have revealed that inactive Rhodopsin GPCRs harbor a conserved binding site for Na+ ions in the center of their transmembrane domain, accessible from the extracellular space. Here, I show that upon the activation of GPCRs, a hydrated channel is formed between the Na+ binding pocket and the cytosol, thereby providing a conduit for Na+ egress to the cytosol. Coupled with the protonation change of D2.50, the Na+ ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. I propose that Na+ ion exchange with the cytosol is a key step in GPCR activation. Further, I hypothesize that this transition locks receptors in long-lived active-state conformations.
Biochemical studies on GPCRs have demonstrated that their basal signalling is allosterically modulated by pH. Here, I show that the global receptor conformation of the δ-opioid receptor and two constitutive mutants is tightly tied to the protonation state of two ultra conserved aspartate residues. I describe a sequential activation pathway linking the Na+ binding site and the D(E)R3.50Y motif to the activation of the receptor.
|Date of Award||2018|
|Sponsors||Biotechnology and Biological Sciences Research Council|
|Supervisor||Ulrich Zachariae (Supervisor) & Timothy Newman (Supervisor)|