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

Acute effect

Chronic effect
 Drugs of Addiction
Chronic effect

Tolerance Dependence
(reduced response (physical need for
to same drug dose) more drug)

Changes in gene expression

 Gene expression
Transcription
Factor
mRNA

RNA
pol.

Promoter Coding Region

 CREB
cAMP Responsive Element Binding-protein

CREB
RNA
Pol.

Promoter CRE Coding Region

Opiate
Activation of CREB

  
GTP

Adenylate Protein CREB

Cyclase Kinase A
(PKA)
P
cAMP CREB

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

Be
n
Alc zyl
oh
ol
 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 CRE Luciferase

•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 Reverse the

Chromatin /DNA 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

Be
n
Alc zyl
oh
ol
 Fig 5
CS

Time (sec)
Tolerance to second
exposure to Benzyl Alcohol
S162 / Y
Measured as number of
drosophila standing after
treatment
Mutations S162 blocks
effect in males (X linked)
but …..
 Fig 5 cont
…… but not in
heterozygous
females.
S 162 / +
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.
(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 ?