WIN55,212-2, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels

Abstract

The coupling of cannabinoid receptors, CB1 and CB2, to G protein-coupled inward rectifier potassium channels, GIRK1 and GIRK2, modulates neuronal excitability in the human brain. The present study established and validated the functional expression in a Xenopus laevis oocyte expression system of CB1 and CB2 receptors, interacting with heteromeric GIRK1/2 channels and a regulator of G protein signaling, RGS4. This ex vivo system enables the discovery of a wide range of ligands interacting orthosterically or allosterically with CB1 and/or CB2 receptors. WIN55,212-2, a non-selective agonist of CB1 and CB2, was used to explore the CB1- or CB2- GIRK1/2-RGS4 signaling cascade. We show that WIN55,212-2 activates CB1 and CB2 at low concentrations whereas at higher concentrations it exerts a direct block of GIRK1/2. This illustrates a dual modulatory function, a feature not described before, which helps to explain the adverse effects induced by WIN55,212-2 in vivo. When comparing the effects with other typical cannabinoids such as Δ9-THC, CBD, CP55,940, and rimonabant, only WIN55,212-2 can significantly block GIRK1/2. Interestingly, the inward rectifier potassium channel, IRK1, a non-G protein-coupled potassium channel important for setting the resting membrane voltage and highly similar to GIRK1 and GIRK2, is not sensitive to WIN55,212-2, Δ9-THC, CBD, CP55,940, or rimonabant. From this, it is concluded that WIN55,212-2 selectively blocks GIRK1/2.

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“…Also, WIN55,212-2, in the presence of HK, induced receptor-dependent inward K+ currents upon I K,basal (IK,WIN) (Figure 1D,E). The EC50 value for WIN55,212.2 to activate CB receptors expressed in oocytes was determined previously in our system (Figure S2) [33]. Additionally, 100 µM OS inhibited 0.3 µM WIN55,212-2-induced inward K + currents (I K,WIN+OS ) in CB-GIRK1/GIRK2-RGS4 coupling systems when they were co-applied (Figure 1F,G).…”

“…Furthermore, the dual modulation of chimeric CB1/2 and GIRK1/GIRK2 channels by WIN55,212-2 is shown in Figure 2C. The WIN55,212-2 concentration-current response curves for activating and blocking CB2 (in blue) and chimeric CB1/2 receptors (in green) are highly overlapping (Figure 2C), indicating that the dual modulatory effect on the chimera and GIRK1/GIRK2 channels by WIN55,212-2 is highly similar to that observed on CB2 receptors and GIRK1/GIRK2 channels, as described before [33].…”

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“…However, in contrast to opioids that inhibit these GABAergic interneurons via pre-and postsynaptic mu opioid receptor activation that modulates inwardly projecting K+ conductance, cannabinoids produce their inhibition of these interneurons strictly via presynaptic CB1s (Vaughan et al, 1999). In several transfected cells or cell lines, activation of CB1s by both endogenous (anandamide) and synthetic (WIN 55,212-2) cannabinoid agonists were found to inhibit neurotransmitter release through Gi/o(βγ) signaling, which inhibits the opening of calcium channels (N&P/Q type) and opens G-protein-gated inward rectifier potassium (GIRK) channels to hyperpolarize the membrane potential of neurons (Mackie et al, 1995;Walsh and Andersen, 2020;Dongchen et al, 2021). Giα, on the other hand, inhibits adenylyl cyclase resulting in a decrease in intracellular cAMP (Howlett et al, 1988).…”

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“…GIRK channels are important regulators of the cellular excitability in the brain and cardiac cells, maintaining the resting membrane potential and regulating the shape and duration of the action potential in excitable cells [5]. In native tissues, GIRK channels are activated by binding of the βγ subunit of Gi/o proteins (Gi/o(βγ)) which disassociates from the Gi/o(α) subunit following the activation of PTX-sensitive G protein-coupled receptors (GPCRs) [6,7]. Thus, the activation of GIRK channels by GPCRs is a crucial part of signal transduction evoked by a diversity of GPCR agonists, including endogenous neurotransmitters such as acetylcholine, dopamine, opioids, serotonin, somatostatin, adenosine, and GABA, as well as exogenous molecules, such as WIN55,212-2 and CP55,940, which are the agonists of cannabinoid receptors [1,2,7,8].…”