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.

Acknowldgements

• Peter O’Day

• Laura Barth

• Bill Roberts

• UO SPUR program

• Sierra Williams & Amy Schilling

• Alice Barkan lab

• LingYa Liao