Voltage-induced structural modifications on M2 muscarinic receptor and their functional implications when interacting with the superagonist iperoxo

It has been reported that muscarinic type-2 receptors (M2R) are voltage sensitive in an agonist-specific manner. In this work, we studied the effects of membrane potential on the interaction of M2R with the superagonist iperoxo (IXO), both functionally (using the activation of the ACh-gated K+ current (I-KACh) in cardiomyocytes) and by molecular dynamics (MD) simulations. We found that IXO activated kAch with remarkable high potency and clear voltage dependence, displaying a larger effect at the hyperpolarized potential. This result is consistent with a greater affinity, as validated by a slower (tau = 14.8 +/- 2.3 s) deactivation kinetics of the IXO-evoked I-KACh than that at the positive voltage (tau = 6.7 +/- 1.2 s). The voltage-dependent M2R-IXO interaction induced I-KACh to exhibit voltage-dependent features of this current, such as the 'relaxation gating' and the modulation of rectification. MD simulations revealed that membrane potential evoked specific conformational changes both at the external access and orthosteric site of M2R that underlie the agonist affinity change provoked by voltage on M2R. Moreover, our experimental data suggest that the 'tyrosine lid' (Y104, Y403, and Y426) is not the previously proposed voltage sensor of M2R. These findings provide an insight into the structural and functional framework of the biased signaling induced by voltage on GPCRs.