Proton-gating of opioid receptors and isoform functionality

The three opioid receptors, mu (mu-OR), delta (delta-OR), and kappa (kappa-OR), are key G-protein-coupled receptors (GPCRs) involved in mediating pain relief. Yet, there has been a lack of understanding regarding how changes in the extracellular pH associated with inflammation affect their signal transduction pathways. This study demonstrates that all opioid receptors act as proton-gated coincidence detectors. A humanized yeast-based platform called DCyFIR, which isolates the pH environment outside the cell from potential confounding factors inside the cell, was employed to examine the activation of opioid receptors over a range of physiologically relevant to pathologically low pH environments. As such, the results show that agonist efficacy decreases with decreasing pH. This indicates that the receptor itself contains one or more protonatable sidechains that act as an intrinsic “pH sensor”. To determine the structural basis of this pH-sensing mechanism, structure-based pHinder calculations were used to identify potential pH sensing residues. Subsequent variant profiling identified the protonation of one conserved aspartic acid residue, D149 (also known as D3.32), located in a region near the binding site of opioid ligands, which, when exposed to acidic pH conditions, resulted in the disruption of the salt bridge necessary for effective docking of opioid ligands with the receptor. Consequently, the receptor lost all ability to be activated by agonists. Further characterization using a variety of functional assays on more than twenty different mu-OR alternative splicing variants indicated that all isoforms that possessed a continuous 7-transmembrane domain retained this proton-sensing capability. These studies collectively provide insight into the structural mechanisms underlying pH-dependent opioid receptor activation and lay the foundation for the design of new generations of selective analgesics that activate opioid receptors only in acidic inflammatory tissue.