Evidencing the role of a conserved polar signaling channel in the activation mechanism of the μ-opioid receptor.

The activity of G protein-coupled receptors has been generally linked to dynamically interconverting structural and functional states and the process of activation was proposed to be controlled by an interconnecting network of conformational switches in the transmembrane domain. However, it is yet to be uncovered how ligands with different extent of functional effect exert their actions. According to our recent hypothesis, the transmission of the external stimulus is accompanied by the shift of macroscopic polarization in the transmembrane domain, furnished by concerted movements of conserved polar amino acids and the rearrangement of polar species. Previously, we have examined the μ-opioid, β(2)-adrenergic and type 1 cannabinoid receptors by performing molecular dynamics simulations. Results revealed correlated dynamics of a polar signaling channel connecting the orthosteric binding pocket and the intracellular G protein-binding surface in all three class A receptors. In the present study, the interplay of this polar signaling channel in the activation mechanism was evidenced by systematic mutation of the channel residues of the μ-opioid receptor. Mutant receptors were analyzed utilizing molecular dynamics simulations and characterized in vitro by means of radioligand receptor binding and G protein stimulation assays. Apart from one exception, all mutants failed to bind the endogenous agonist endomorphin-2 and to stimulate the G(i) protein complex. Furthermore, mutation results confirmed allosteric connection between the binding pocket and the intracellular surface. The strong association and optimal bioactive orientation of the bound agonist was found to be crucial for the initiation of correlated motions and consequent signaling.