Transcriptional mechanisms that produce BK channel-dependent 
drug tolerance and dependence

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Li, Xiaolei, Ph. D.

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Tolerance to anesthetic drugs is mediated partially by homeostatic mechanisms that attempt to restore normal neural excitability. The BK-type Ca2+-activated K+ channel, encoded by the slo gene, plays an important role in this neural adaptation. In Drosophila, a single sedative dose of the organic solvent anesthetic benzyl alcohol induces dynamic spatiotemporal changes in histone H4 acetylation across the slo regulatory region and leads to slo induction and tolerance. Mutations ablating the expression of slo also block the acquisition of tolerance, whereas activating the expression of a slo transgene results in resistance to drug sedation. Moreover, artificially inducing histone acetylation with the histone deacetylase inhibitor causes similar acetylation changes, slo induction, and functional tolerance to the drug. Histone acetylation changes occur over two highly conserved non-coding DNA elements, 6b and 55b, of the slo control region. To investigate the function of these two elements, I generated individual knockout mutants by gene targeting. Both knockout alleles are backcrossed into the CS wild type background. The 6b element seems to repress slo induction after drug sedation, because the 6b knockout allele overreacts to the drug. Compared to the wild type, 6b knockout allele shows a much greater slo message induction after drug sedation, it also displays stronger enhancements in seizure susceptibility and following frequency. In addition, the 6b deletion causes a persistent tolerance for at least a month, while tolerance only lasts about 10 days in wild type flies. My investigation also indicates that the 55b element limits basal slo expression in muscle. Finally, to investigate if the particular histone acetylation spikes are required for drug-induced slo induction and tolerance, I tether the histone-modifying enzymes, HDAC or HAT, to the 6b and 55b DNA elements, respectively. I observe that the positioning of an HDAC on these two elements blocks drug-induced slo induction and the development of tolerance. Therefore, histone acetylation across slo control region is required for the activation of slo and the acquisition of tolerance.



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