Transcriptional mechanisms that produce BK channel-dependent drug tolerance

Abstract

Tolerance to drugs that affect neural activity is mediated, in part, by adaptive mechanisms that attempt to restore normal neural excitability. Changes in the expression of ion channel genes are thought to play an important role in this neural adaptation. The slowpoke (slo) gene encodes the pore-forming subunit of BK-type Ca2+-activated K+ channels which regulate many aspects of neural activity. In Drosophila, behavioral tolerance induced by a single anesthetic sedation has been associated with the induction of slo expression in the nervous system. Regulation of gene expression is achieved by a complex array of molecular mechanisms including histone modification, chromatin decondensation, and recruitment of transcription factors and co-factors to specific DNA elements. In this study, I investigate the production of specific histone modifications at slo promoters caused by drug sedation, as well as the roles of specific transcription factors on slo induction and the development of drug tolerance. Using the chromatin immunoprecipitation assay followed by real-time PCR, I show that a single brief sedation with the anesthetic benzyl alcohol generates a specific spatiotemporal pattern of histone modification across the slo promoter region. The pattern of histone H4 acetylation is correlated with the induction of slo messenger RNA. Artificially inducing histone acetylation, utilizing a histone deacetylase inhibitor, yields a similar change in histone H4 acetylation, up-regulates slo expression, and phenocopies tolerance in a slo-dependent manner. Sequence analysis has identified several evolutionarily conserved regions in slo promoters. These contain DNA elements that could be recognized by transcription factors such as CREB, AP-1 and HSF. CREB transcription factors, which can recruit CBP and cause histone acetylation, are involved in the development of tolerance in both mammals and flies. In this study, CREB function is linked to the sedation-induced up-regulation of the slo gene and to drug tolerance. Sedation with the anesthetic benzyl alcohol down regulates the mRNA level of the CREB repressor splice variant but does not affect the level of the CREB activator splice variant. The down regulation of the CREB repressor increases CREB target gene expression. Chromatin immunoprecipitation assays with anti-CREB antibodies indicate that sedation with benzyl alcohol increases the occupancy of CREB within the slo transcriptional control region. In addition, a loss-of-function mutation in CREB and an inducible dominant negative CREB transgene block both sedation-induced slo induction and the ability of animals to acquire tolerance after anesthetic sedation. The induction of a dominant negative CREB transgene also blocks the formation of the early histone acetylation peak, caused by benzyl alcohol sedation, within the slo promoter region. These findings support the hypothesis that drug sedation activates the CREB signaling pathway, recruits CREB to the slo promoter region, and that CREB induces histone acetylation by recruiting CBP. Histone acetylation opens the chromatin structure at the slo promoter region and facilitates gene transcription. Increased expression of slo channels are predicted to enhance the capacity of neurons for repetitive activity, which may speed the recovery of flies from sedation.