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WIN55,212-2, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels.
An D
,
Peigneur S
,
Tytgat J
.
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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.
Figure 1. Activation of the CB-GIRK1/2-RGS4 coupling in Xenopus oocytes. (A) Oocytes co-injected with CB1, GIRK1/2, and RGS4, and (B) oocytes co-injected with CB2, GIRK1/2, and RGS4 were voltage-clamped at −90 mV. IK,basal was enhanced by exchanging HK to HK plus 1 μM WIN55,212-2. Typical IK,basal and IK,WIN were recorded from three to six different oocytes (n = 3–6).
Figure 2. Activation of inward rectification of GIRK1/2 in the CB-GIRK1/2-RGS4 coupling in Xenopus oocytes. (A) Oocytes co-injected with CB1, GIRK1/2, and RGS4 cRNAs, and (B) oocytes co-injected with CB2, GIRK1/2, and RGS4 cRNAs were subjected to a 1-s voltage ramp protocol from −150 to +60 mV from a holding potential of −20 mV. Inward IK,basal was enhanced by exchanging HK to HK plus 1 μM WIN55,212-2. Typical IK,basal and IK,WIN were recorded from three to six different oocytes.
Figure 3. Concentration-response curve of WIN55,212-2 for CB-GIRK1/2-RGS4 coupling and GIRK1/2. Oocytes were co-injected with CB, GIRK1/2, and RGS4, or GIRK1/2 cRNAs. Inward K+ current enhancement and reduction were produced on the application of a range of different concentrations of WIN55,212-2 in the presence of HK. The positive percentage represents enhancement and the negative percentage represents reduction. Each data point is the mean ± standard deviation (SD) of three determinations from two or three batches of oocytes.
Figure 4. Reduction of inward K+ currents in the CB-GIRK1/2-RGS4 coupling and GIRK1/2 in Xenopus oocytes. (A) Oocytes co-injected with CB1, GIRK1/2, and RGS4 cRNAs, (B) oocytes co-injected with CB2, GIRK1/2, and RGS4 cRNAs, and (C) oocytes injected with GIRK1/2 cRNAs were subjected to a 1-s voltage ramp protocol from −150 to +60 mV from a holding potential of −20 mV. Inward IK,basal was strongly reduced by exchanging HK to HK plus ≈ 100 μM WIN55,212-2. Typical IK,basal and IK,WIN were recorded from three to six different oocytes.
Figure 5. The best fit for concentration–current activation curve of WIN55,212-2 for CB-GIRK-RGS4 coupling. Oocytes were co-injected with CB, GIRK1/2, and RGS4 cRNAs. (A) CB1 activation and (B) CB2 activation were produced on the application of a range of different concentrations of WIN55,212-2 in the presence of HK. Each data point is the mean ± SD of three determinations from two or three batches of oocytes.
Figure 7. Comparison of effects of WIN55,212-2, ∆9-THC, CBD, CP55,940, and rimonabant on GIRK1/2. ≈100 μM of WIN55,212-2 and 100 μM of CBD, CP55,940, and rimonabant reduced inward K+ currents, while 100 μM ∆9-THC slightly enhanced inward K+ currents. The positive percentage represents enhancement and the negative percentage represents reduction. Statistical comparisons between the various experimental groups were performed by Dunnett’s multiple comparisons test of one-way ANOVA where p < 0.05 was considered significant. ****, p < 0.0001 vs. percentage for WIN55,212-2. Each data point is the mean ± SD of three determinations from two or three batches of oocytes
Figure 6. Structures of WIN55,212-2, ∆9-THC, CBD, CP55,940, and rimonabant, and changes of inward K+ currents through GIRK1/2 and IRK1 produced by these cannabinoids. Structures of WIN55,212-2, ∆9-THC, CBD, CP55,940, and rimonabant are shown in (a) column. (b) Oocytes injected with GIRK1/2 and (c) oocytes injected with IRK1 were subjected to a 1-s voltage ramp protocol from −150 to +60 mV from a holding potential of −20 mV. Current enhancement or reduction was produced on the application of ≈100 μM of (A) WIN55,212-2, or 100 μM of (B) ∆9-THC, (C) CBD, (D) CP55,940, and (E) rimonabant. Typical IK,basal and IK,compound were recorded from three to five different oocytes.
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