CREB regulation of BK channel gene expression

underlies rapid drug tolerance

Y. Wang†,‡, A. Ghezzi†, J. C. P. Yin§,¶

and N. S. AtkinsonGenes, Brain and Behavior (2009)

Drugs of AddictionDrugDrug

Acute effect Acute effect

DrugDrug

Acute effect Acute effect

DrugDrug

Acute effect Acute effect

Chronic effectChronic effect

Drugs of Addiction

Chronic effectChronic effect

Dependence(physical need for more drug)

Dependence(physical need for more drug)

Tolerance(reduced response to same drug dose)

Tolerance(reduced response to same drug dose)

Changes in gene expression Changes in gene expression

Gene expression

PromoterPromoter Coding RegionCoding Region

TranscriptionFactor

TranscriptionFactor

RNA pol.RNA pol.

mRNA

CREBcAMP Responsive Element Binding-protein

PromoterPromoter Coding RegionCoding RegionCRECRE

CREBCREB

RNAPol.RNAPol.

Activation of CREB

AdenylateCyclase

AdenylateCyclase

GTP

Protein Kinase A

(PKA)

Protein Kinase A

(PKA)

CREBCREB

PromoterPromoter CodingCodingCRECRE

cAMPcAMP CREBCREBPP

Opiate

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

BenzylAlcohol

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

PromoterPromoter LuciferaseLuciferaseCRECRE CRECRE

•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

Crosslink Chromatin /DNA

Sonicate to fraction DNA

Immunoprecipitate with P-CREB antibodies

Reverse the Crosslink

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.

This shows that benzyl alcohol-induced slo expression is co-ordinated by the dCREB2 transcription factor because dCREB2S162 is dominant negative on dCREB2

S162 CS male

Recovery time from anesthesia

BenzylAlcohol

Fig 5

Tolerance to second exposure to Benzyl AlcoholMeasured as number of drosophila standing after treatment Mutations S162 blocks effect in males (X linked) but …..

Time (sec)

CS

S162 / Y

Fig 5 cont

…… 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

Slo

Ca++Na+K+

K+

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 ?