CB1 receptor signaling regulates Cx43 hemichannel trafficking and phosphorylation in astrocytes: implications for gliotransmission

Background: Connexin 43 (Cx43), the most abundant connexin in the central nervous system, is predominantly expressed in astrocytes, where it mediates intercellular communication and energy exchange via gap junctions and hemichannels. Although Cx43 hemichannels are critically involved in neuropathological injury, the regulatory mechanisms controlling their expression and activity remain poorly understood. Previous studies indicate that cannabinoid receptor type 1 (CB1R) activation enhances Cx43 hemichannel opening by elevating intracellular calcium levels in astrocytes, thereby modulating synaptic transmission and neural network plasticity. Methods: This study aimed to investigated whether the CB1R pathway regulates Cx43 hemichannel expression, intracellular trafficking, phosphorylation, and functional activity in astrocytes. The hum an astrocyte cell line U251 was cultured in vitro. Western blotting was performed to assess total and membrane-associated Cx43 protein levels following treatment with the CB1R agonist Bay59-3074 or the CB1R antagonist JD-5037. Membrane permeability was evaluated via dye uptake assays, while gliotransmitter release (ATP and GABA) and intracellular calcium levels were measured using ELISA kits and the fluorescent probe Fluo-3 AM, respectively. Results: Our findings unveiled that neither Bay59-3074 nor JD-5037 altered total Cx43 protein levels. However, the CB1R agonist group exhibited reduced total Cx43 phosphorylation (p-Cx43/Cx43 ratio) but increased membrane Cx43 localization, whereas the antagonist group showed elevated phosphorylation and decreased membrane Cx43. Dye uptake efficiency significantly increased in the agonist-treated group, indicating enhanced hemichannel activity. Lentivirus-mediated Cx43 knockdown attenuated CB1R agonist-induced calcium influx and gliotransmitter release (ATP and GABA), confirming Cx43 dependency. Conclusion: These results demonstrate that the CB1R pathway regulates Cx43 hemichannel membrane trafficking and phosphorylation, thereby modulating astrocytic membrane permeability and gliotransmitter release. Targeting astrocytic Cx43 hemichannels via CB1R signaling may represent a novel therapeutic strategy for neurological disorders associated with dysregulated intercellular communication.