Transcript CREB regulation of BK channel gene expression underlies

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
(reduced response
to same drug dose)
Dependence
(physical need for
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
a b g
GTP
Adenylate
Cyclase
CREB
Protein
Kinase A
(PKA)
P
cAMP
Promoter
CRE
CREB
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
• 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
Chromatin /DNA
Sonicate to
fraction DNA
http://www.millipore.com/cellbiology/cb3/chromatinip
Reverse the
Crosslink
Detect
sequences by
Real time PCR
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 coordinated by the dCREB2 transcription factor because
dCREB2S162 is dominant negative on dCREB2
Recovery time from anesthesia
Fig 5
CS
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
…… but not in
heterozygous
females.
i.e. [S162]-d-CREB2
does not works in
negative dominant
manner
demonstrating that
phosphorylated dCREB2 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 voltagegated 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 ?