Download Calretinin

Is Phosphorylation of
Calretinin a Mechanism for
Ca2+ Regulation at
Synapses?
Erika Marulanda
O’Day Lab
SPUR 2005
Neuronal Connections
•Neuron receives stimulus
•Action Potential: Voltagegated channels allow Ca2+
into the cell.
•Neurotransmitters released
into the synaptic cleft & bind
to receptors on post-synaptic
cell
•Cell needs only a small
amount of Ca 2+ to signal
transmitter release
Calretinin plays a role in regulating
the amount of Ca2+ in pre-synaptic
cell.
• Ca2+ binding protein
present in the neurons of
most vertebrates
• Functions as a Ca2+ buffer
• 6 EF hands but only four
or five of appear to be
functional and suitable for
Ca2+ binding/buffering
• The EF-hand Ca2+ binding
site consists of an α-helix,
a loop, and another αhelix
EF Hands
• The Ca2+ ion binds to the loop
connecting the two helices.
• In this loop, there are amino
acid residues with negatively
charged oxygen atoms, which
attract the positively charged
calcium ion
• When Ca2+ enters the cell
Calretinin binds about 99% of it.
• Calretinin releases Ca2+ after
diffusing away to areas of low
Ca2+.
The Big Question: How is binding regulated?
Ca2+ binding by Calretinin is
regulated by phosphorylation
• Phosphorylation is
addition of a phosphate
group
• Molecular “on- off” switch
• Alter shape and function
• changes how the protein
interacts with other
proteins or signal
molecules, such as Ca2+
Insert picture of
protein
phosphorylation
diagram scheme
The Experiment
• Goal: to determine if Calretinin is phosphorylated. This could
be a mechanism for regulating Ca2+ binding by Calretinin
• Animal model: Zebrafish
– Calretinin is found in zebrafish retina
– Retina is organ that
• Protocol
– Immunoprecipitation
– Incubation in radioactive orthophosphate
– Western Blot
http://webvision.umh.es/webvision/sr
etina.html
Phosphorylation Sites
– 5 potential sites for
phosphorylation
– 2 of the sites are located
within an EF hand
– Phosphorylation may
change the conformation
of the EF hands of
Calretinin
– this may cause a change
in the Ca 2+ binding sites,
& thus, be a means of
regulation.
Experimental Design: Incubation
and Purification
• Control: No retina, no calretinin
• Incubate retina in orthophosphate: Use
of radioactivity provides a way to
visualize phosphorylation
• Homogenize the retina
• Purify Calretinin using
Immunoprecipitation
– Staph A beads bind to calretinin
– Polyclonal Calretinin antibody binds
to beads
– All other proteins remain in solution
Calretinin
Beads
Experimental Design: Visualize
Radioactivity
•
•
•
•
Run purified Calretinin (and control) on gel
Transfer protein from gel to membrane
Expose to x-ray film and develop
Any band with radioactivity is visible on film.
But how will you know if visible band is Calretinin?
Experimental Design: Western
Blot
• Antibodies bind to proteins
very specifically
• 10 Ab: mouse anti-CR will
bind to Calretinin
• 20 Ab: goat-anti-mouse will
bind to 10 Ab
• Use chemiluminscence to
detect 20 Ab.
• Develop x-ray film
• Only band with Calretinin
should be labeled.
http://probes.invitrogen.com/handbo
ok/images/g001474.gif
Compare films to see if Calretinin is in the same place
Results
• The Control Lane had
no Calretinin but there
was significant labeling
• Possible interference
• Cannot be sure that
labeled band in protein
lane indeed contains
calretinin
What could be the source of Interference?
Possible Source of Interference
o A polyclonal Ab was used in
purifying calretinin
o The polyclonal Ab was
present in protein and noprotein lanes
o It always travels to same
location, and this location
may be where calretinin is
located.
o The secondary Ab used in
the western blot may be
binding to the polyclonal Ab
as well as to the 1o Ab.
o The labeling occurs in the
same spot that calretinin
would be labeled.
Conclusions
• Goal: to determine if Calretinin is phosphorylated. This could be a
mechanism for regulating Ca2+ binding by Calretinin
• Results: Incomplete: we have not yet determined calretinin labeling
in westerns.
• The polyclonal Ab used to purify calretinin caused interference in
the western blot.
• Determining whether CR phosphorylation is a mechanism for
regulating its Ca2+ binding will require: first labeling CR
unambiguously; and second determining by autoradiography
whether CR is phosphorylated under a variety of conditions. The
present work represents a contribution to investigating the larger
question.
Further Experiments
• Need a strategy to eliminate the polyclonal
Antibody signal interference.
• Possibility: Use a different secondary
antibody in Western blot. In experiments
after I left, a secondary antibody was used
successfully which labels only native
IgG’s.
Acknowldgements
•
•
•
•
•
•
•
Peter O’Day
Laura Barth
Bill Roberts
UO SPUR program
Sierra Williams & Amy Schilling
Alice Barkan lab
LingYa Liao