is phosphorylation of calretinin a mechanism for ca 2+ regulation at synapses? erika marulanda...
TRANSCRIPT
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: Voltage- gated 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 α-helix
• 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+.
EF Hands
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/sretina.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
BeadsCalretinin
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/handbook/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 no- protein 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.