In this article, we characterized protein-protein interactions within a complex involved in opioid analgesia which is formed by delta opioid receptors (DORs), heterotrimeric G protein (Gαoβ1γ2) and their effector, the G protein-gated inwardly rectifying potassium channel (GIRK/Kir3). It is well established that sustained DOR stimulation by an agonist triggers a series of adaptive changes that reduce receptor ability to signal and this desensitization may contribute to analgesic tolerance.
Although there is considerable information of how desensitization reduces receptor ability to interact and activate the G protein, much less is known on how desensitization modifies the channel standing in the complex. We therefore focused on how interactions between the channel and other complex components were modified by sustained receptor activation.
Our results show that DORs, G protein and Kir3 channels form a constitutive complex at the plasma membrane. This complex undergoes rapid conformational rearrangements upon acute DOR stimulation and maintains its integrity over more prolonged periods of receptor activation. During this time, the DOR/G protein/Kir3 complex undergoes additional conformational changes imposed by βarrestin 2 (βarr2) recruitment and association with receptors and channels. This interaction not only induces DOR removal from the membrane but also that of the channel. Both signaling partners are concomitantly internalized via a clathrin and dynamin-dependent mechanism.
Conclusion: Taken together, these data show that DORs and Kir3 channels form a constitutive complex which is recognized and internalized as a signaling unit by βarr2.
Contribution to the advancement of knowledge: Kir3 channels removal from the membrane represents an additional level of regulation of opioid receptor signaling that had not been previously described. Moreover, given active Kir3 channels participation in opioid analgesia, their removal from the membrane may constitute an additional and powerful mechanism of tolerance. Thus, it is reasonable to expect that development of DOR ligands that activate the channel but could prevent complex interaction with βarr2 could lead to the production of opioid analgesics that preserve their therapeutic efficacy.
– Kir3.1/3.2 channels, G proteins and DORs form a complex.
– The complex maintains its integrity over prolonged periods of receptor stimulation.
– βarr2 is recruited to DORs and channels mediating their internalization as a unit.
– DOR-Kir3 channel internalization is clathrin/dynamin dependent.
Figure Legend. Mechanism of analgesia induced by Kir3 channels at the synaptic cleft. When released into the synaptic cleft, neurotransmitters such as endogenous opioids (pink) activate the DOR receptor (red) of the postsynaptic neuron, which in turn activate the Kir3 channel (green). Activation of Kir3 channels produces hyperpolarization at the postsynaptic membrane thereby reducing the transmission of nociceptive impulses.
Note: modified figure from figure 1 Front Cell Neurosci. 2014 Jul 8;8:186 and figure 4 suppl Cell Mol Life Sci. 2015 Sep;72(18):3543-57.
Citation: Effect of collagen-glycosaminoglycan scaffold pore size on matrix mineralization and cellular behavior in different cell types. Karim Nagi, Iness Charfi and Graciela Pineyro. Cell Mol Life Sci. 2015 Sep;72(18):3543-57.