1. 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)

2. Drugs of Addiction Drug Acute effect Drug Acute effect Drug
Chronic effect

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

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

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

6. 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

7. 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

8. 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

9. Basic technique
Benzyl
Alcohol

10. 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

11. 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 Dec 7;101(49): Epub 2004 Nov 29.slo K(+) channel gene regulation mediates rapid drug tolerance.Ghezzi A, Al-Hasan YM, Larios LE, Bohm RA, Atkinson NS.

12. 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.

13. 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)

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

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

16. Fig 3a map of Slo promoter
CO b cre S b cre2

17. Chromatin Immunoprecipitation
Immunoprecipitate with P-CREB antibodies
Crosslink Chromatin /DNA
Reverse the Crosslink
Sonicate to fraction DNA
Detect sequences by
Real time PCR

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

20. Fig 4: The dCREB2S162 mutation blocks induction of slo by benzyl alcohol (BA) sedation.
S 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

21. Recovery time from anesthesia
Benzyl
Alcohol

22. 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

23. 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 / +

25. (histone acetylator)

26. 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

27. 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.

28. 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 ?