It has been suggested that drug tolerance represents a form of learning and memory, but this has not been experimentally established at the molecular level. We show that a component of alcohol molecular tolerance (channel internalization) from rat hippocampal neurons requires protein synthesis, in common with other forms of learning and memory. We identify -catenin as a primary necessary protein.Alcohol increases -catenin, and blocking accumulation of -catenin blocks alcohol-induced internalization in these neurons. In transfected HEK293 cells, suppression of Wnt/-catenin signaling blocks ethanol-induced internalization. Conversely, activation of Wnt/-catenin reduces BK current density. A point mutation in a putative glycogen synthase kinase phosophorylation site within the S10 region of BK blocks internalization, suggesting that Wnt/-catenin directly regulates alcohol-induced BK internalization via glycogen synthase kinase phosphorylation. These findings establish de novo protein synthesis and Wnt/-catenin signaling as critical in mediating a persistent form of BK molecular alcohol tolerance establishing a commonality with other forms of long-term plasticity.
Alcohol tolerance development and retention has long been associated to neural mechanisms related to learning and memory. We have found a persistent component of alcohol molecular tolerance; internalization of BK channels, in both striatal and hippocampus neurons. It is further characterized by increases in β‐catenin in response to physiologically relevant concentrations of alcohol, which are necessary for BK channel internalization. Accumulation of β‐catenin was impaired in the presence of general protein synthesis inhibitors cyclohexamide and emetine, which further blocked ethanol‐induced BK channel internalization in heterologous expression system. Interestingly, BK channel dissociation from lipid raft fractions in response to ethanol exposure was not significantly affected by protein synthesis inhibition. Translational regulation of EtOH was evaluated via tandem mass spectrometry (MS/MS) and Western Blot analysis of de novo synthesized proteins in heterologously expressed hSloα HEK293 preparations. Results indicated that β‐catenin is increased 2.5 fold in response to alcohol compared within a subset of over seven hundred proteins assayed. Transcriptional regulation of EtOH in striatal tissue has been further studied suggesting changes in miRNA profile in response to duration of EtOH exposure. These findings suggest the canonical Wnt/β‐catenin signaling pathway plays a critical role in mediating a persistent form of BK channel molecular alcohol tolerance regulating BK channel surface distribution, and potentially mediating transcriptional regulation in response to EtOH.Support or Funding InformationThis work was supported by the COBRE project grant 5P20GM103642 (CV‐M), Imaging core at the Institute of Neurobiology: NSF Grant DBI‐0115825 and DoD Grant 52680‐LS‐IS, Research infrastructure support was provided in part by a grant from the National Institute of Minority Health and Health Disparities (G12MD 007600), and USDE Title V Cooperative Agreement grant P031S130068 (JOG).
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