CREB regulation of BK channel gene expression underlies rapid drug tolerance Y. Wang†,‡, A. Ghezzi†, J. C. P. Yin§,¶ and N. S. Atkinson Genes, Brain and Behavior (2009)

Drugs of Addiction Drug Acute effect Drug Acute effect Drug Chronic effect

Drugs of Addiction Chronic effect Tolerance (reduced response to same drug dose) Dependence (physical need for more drug) Changes in gene expression

Gene expression Promoter Coding Region Transcription Factor mRNA RNA pol. Promoter Coding Region

CREB cAMP Responsive Element Binding-protein RNA Pol. Promoter CRE Coding Region

Activation of CREB a b g Adenylate Cyclase CREB Protein Kinase A (PKA) Opiate Activation of CREB a GTP b g Adenylate Cyclase CREB Protein Kinase A (PKA) CREB P cAMP Promoter CRE Coding

BKCa channel ( Slo ) Ca++ activated, voltage dependent K+ channel Ubiquitously distributed in exitable and non excitable cells Involved in action potential, neurotransmitter release etc. Differ from other K+ channels due to their sensitivity to Ca++ Feedback regulator: hyperpolarise cell after Ca++ entry

CREB in drosophila(fruit fly) Two genes dCREB-A and dCREB-2 dCREB-2 believed to be homoloug to mammalian CREB dCREB-2 has role in the production of circadian rhythms, learning, memory and sexual behavior . Splice variants found Most have kinase-inducible domain (Ser133) dCREB-2b splice variant is negative regulator of dCREB-2

Basic technique Benzyl Alcohol

Benzyl Alcohol Produced by many plants and is commonly found in fruits, teas and a variety of essential oils including jasmine, hyacinth and ylang-ylang Benzyl alcohol is used as a general solvent for inks, paints, lacquers, and epoxy resin coatings. Organic solvent have been used both as anesthetic and drug of abuse

Previous finding by same authors Single sedation with benzyl alcohol: Changes in expression of BK channel Induction of tolerance Hyper-acetylation of histone H4 protein within Slo promoter (exposes DNA) CREB binds to Slo promoter Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17276-81. Epub 2004 Nov 29.slo K(+) channel gene regulation mediates rapid drug tolerance.Ghezzi A, Al-Hasan YM, Larios LE, Bohm RA, Atkinson NS.

Aim of this paper Investigate the mechanism of activation of Slo promoter by benzyl alcohol (hypothesis: CREB) Demonstrate that CREB induced expression of Slo channels is associated with tolerance.

Fig 1 Benzyl alcohol (BA) sedation enhances dCREB-A and reduces dCREB2 repressor splice variant (dCREB2-b) mRNA abundance. Method: real-time Reverse transcriptase PCR (Cyp1 internal standard)

CRE-luciferase assay Trangenic fly with CRE reporter gene Promoter CRE Treat with Benzyl alcohol Separate drosophila heads Measure luciferase activity in luminometer

Figure 2: Positive regulation by CREB increases after benzyl alcohol (BA) sedation.

Fig 3a map of Slo promoter CO 6b cre1 S2 55b cre2

Chromatin Immunoprecipitation Immunoprecipitate with P-CREB antibodies Crosslink Chromatin /DNA Reverse the Crosslink Sonicate to fraction DNA Detect sequences by Real time PCR http://www.millipore.com/cellbiology/cb3/chromatinip

Fig 3 Demonstrates the cre 1 and cre 2 are occupied by P-CREB following stimulation with Benzyl-alcohol.

Fig 4: The dCREB2S162 mutation blocks induction of slo by benzyl alcohol (BA) sedation. S162 CS male This shows that benzyl alcohol-induced slo expression is co-ordinated by the dCREB2 transcription factor because dCREB2S162 is dominant negative on dCREB2

Recovery time from anesthesia Benzyl Alcohol

Fig 5 CS Tolerance to second exposure to Benzyl Alcohol Time (sec) Tolerance to second exposure to Benzyl Alcohol Measured as number of drosophila standing after treatment Mutations S162 blocks effect in males (X linked) but ….. S162 / Y

Fig 5 cont S 162 / + …… but not in heterozygous females. i.e. [S162]-d-CREB2 does not works in negative dominant manner demonstrating that phosphorylated d-CREB2 is necessary to induce tolerance to Benzyl alcohol. S 162 / +

(histone acetylator)

Proposed mechanism of tolerance Stimulation of dCREB2 leads to acetylation of slo promoter DNA Upregulation of BK channels enhances the capacity for repetitive firing by limiting the inactivation of voltage-gated Ca2++ and Na+ channels and/or by preventing the activation of other K+ channels K+ Na+ Ca++ K+ Slo

Key results Benzyl increases dCREB-2 dCREB-2 stimulates BKCa /slo expression BKCa /slo expression is associated with tolerance All of the components and mechanism described in drosophila also exist in mammals.

Questions to be asked What is the mechanism of increase dCREB-2 mRNA ? (regulation via phosphorylation vs gene expression) What other transcription factors are activated by benzyl alcohol ? What is the exact relationship between the increase BKCa /slo and tolerance ?