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Kir3 channels undergo arrestin-dependant internalization following delta opioid receptor activation

Significance Statement

Protein-protein interactions are of great importance for virtually all biological processes and whether transient or stable, they support the formation of multimeric complexes. Monitoring such interactions may help us characterize signaling and trafficking behaviours of these complexes and sometimes allows us to elucidate new signaling pathways for a known protein. A better and detailed knowledge of these different aspects of complex function is essential not only to understand the majority of physiological processes but also for the development of new therapeutic ligands.

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.Kir3 channels undergo arrestin-dependant internalization following delta opioid receptor activation-Global Medical Discovery

About The Author

Dr. Karim Nagi received his Bachelor’s degree in Biology from the Lebanese University, Tripoli, Lebanon (2005-2008). He then moved to Canada where he completed one year in basic research in Molecular Cardiology and Genetics at Sacré-Cœur Hospital’s Research Center, Montreal, Canada. In 2010, he entered the MSc program at the Department of Pharmacology, University of Montreal under the supervision of Prof. Pineyro at Sainte-Justine Hospital Research Center. By the end of his first year, he was offered accelerated switch to the PhD program which he completed in 2015. During this time Dr Nagi applied a variety of approaches in biochemistry and neuroscience to investigate the network properties of GPCR signaling with particular focus in the analgesic actions of opioid receptor ligands and their potential to induce tolerance.

Dr. Nagi has authored 7 publications in peer-reviewed journals and 50+ presentations. In addition, he received eight awards for the best oral and poster presentations in scientific conferences, two travel awards to international congresses and his studies were supported by a number of fellowship awards including CHU Sainte-Justine and Foundation of Stars fellowship, fellowship from the Department of Pharmacology and a fellowship from the Faculty of Graduate and Postdoctoral Studies, University of Montreal. He was also granted a Recognition Award for the Best Scientific Contribution of the year (2014-2015) among students in the Department of Pharmacology.

After completing graduate studies, Dr. Nagi continued his training as a postdoctoral fellow in the Department of Cellular Biology, Duke University, Durham, USA under the supervision of Prof. Marc G. Caron. His current research focuses on characterizing different biased receptors signaling and regulation.

Working at these different institutions with a world-renowned reputation in pharmacological research, Dr. Nagi has developed expertise in GPCRs pharmacology, BRET-based biosensor development for drug screening, molecular biology and biased signaling.

About The Author

Graciela Pineyro, MD, Ph.D. 

Prof. Graciela Pineyro is a Full Professor at the Department of Psychiatry, University of Montreal, Montreal, Canada. In 1991, she received her medical degree with specialty in Pharmacology from the Faculty of Medicine, National University, Uruguay. She then moved to Canada where she obtained a Ph.D. in Neurosciences from McGill University, Montreal, Canada (1997) followed by postdoctoral training in molecular pharmacology in the Department of Biochemistry, University of Montréal (1997-2001). During her career she was supported by different fellowships and awards including McGill Major Fellowships (Canada), Fogarty-NIH International Fellowship (USA) and Postdoctoral Fellowships from Medical Research Council of Canada and Heart and Stroke Foundation of Canada.

Today, she is head of a pharmacology laboratory with research focus on molecular determinants of analgesic efficacy of opioids, as well as cellular and molecular bases of analgesic tolerance. She has substantially contributed to the notion of biased signaling showing that delta opioid receptors adopt ligand-specific conformations with distinct signaling and trafficking properties. Insights from her research have provided the basis for the rational development of novel opioid analgesics with a reduced side effects profile.

Prof. Pineyro has authored 37 peer-reviewed publications, 7 book chapters and 100+ presentations, and holds 2 licensed patents.

As an independent investigator, she has received the New Investigator Award from Fond de Recherche en Santé du Québec and her research has been continuously funded by Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada.

About The Author

Iness Charfi, MSc 

Iness Charfi received her Bachelor in Pharmacy in 2008 at Monastir University (Tunisia). In 2009, she joined Prof. Graciela Pineyro’s Lab as an MSc student in Neuropsychopharmacology at the Sainte-Justine Hospital Research Center, through the Department of Pharmacology, University of Montreal (Canada). After graduating in 2012, she started a PhD in the same laboratory. Throughout her training, she focused on the mechanistic understanding of the molecular basis of delta opioid receptor post-endocytic trafficking, in order to better understand the development of analgesic tolerance to opioids. Iness has been awarded a number of prizes and fellowships, including two presentation awards for best oral and poster presentations at scientific meetings. Her fellowship awards include a CHU Sainte-Justine and Fondation of Stars fellowship, a recruitment fellowship from the Department of Pharmacology, University of Montreal and a fellowship award from the FRSQ.


Journal Reference

Cell Mol Life Sci. 2015 Sep;72(18):3543-57.

Karim Nagi1,2, Iness Charfi1,2 and Graciela Pineyro1,2,3

Show Affiliations
  1. Sainte-Justine Hospital Research Center, Montreal, Quebec, H3T 1C5, Canada.
  2. Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4, Canada.
  3. Department of Psychiatry, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4, Canada.


Kir3 channels control excitability in the nervous system and the heart. Their surface expression is strictly regulated but mechanisms responsible for channel removal from the membrane remain incompletely understood. Using transfected cells, we show that Kir3.1/3.2 channels and delta opioid receptors (DORs) associate in a complex which persists during receptor activation, behaving as a scaffold that allows beta-arrestin (βarr) to interact with both signaling partners. This organization favored co-internalization of DORs and Kir3 channels in a βarr-dependent manner via a clathrin/dynamin-mediated endocytic path. Taken together, these findings identify a new way of modulating Kir3 channel availability at the membrane and assign a putatively novel role for βarrs in regulating canonical effectors for G protein-coupled receptors.

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