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.