1. Is Phosphorylation of Calretinin a Mechanism for Ca 2+ Regulation at Synapses? Erika Marulanda O’Day Lab SPUR 2005

2. Neuronal Connections Neuron receives stimulus Action Potential: Voltage- gated channels allow Ca 2+ 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

3. Calretinin plays a role in regulating the amount of Ca 2+ in pre-synaptic cell. Ca 2+ binding protein present in the neurons of most vertebrates Functions as a Ca 2+ buffer 6 EF hands but only four or five of appear to be functional and suitable for Ca 2+ binding/buffering The EF-hand Ca 2+ binding site consists of an α-helix, a loop, and another α- helix

4. The Ca 2+ 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 Ca 2+ enters the cell Calretinin binds about 99% of it. Calretinin releases Ca 2+ after diffusing away to areas of low Ca 2+. EF Hands The Big Question: How is binding regulated?

5. Ca 2+ 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 Ca 2+ Insert picture of protein phosphorylation diagram scheme

6. The Experiment Goal: to determine if Calretinin is phosphorylated. This could be a mechanism for regulating Ca 2+ 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

7. 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.

8. 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 Beads Calretinin

9. 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?

10. Experimental Design: Western Blot Antibodies bind to proteins very specifically 1 0 Ab: mouse anti-CR will bind to Calretinin 2 0 Ab: goat-anti-mouse will bind to 1 0 Ab Use chemiluminscence to detect 2 0 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

11. 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?

12. 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 1 o Ab. o The labeling occurs in the same spot that calretinin would be labeled.

13. Conclusions Goal: to determine if Calretinin is phosphorylated. This could be a mechanism for regulating Ca 2+ 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 Ca 2+ 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.

14. 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.

15. Acknowldgements Peter O’Day Laura Barth Bill Roberts UO SPUR program Sierra Williams & Amy Schilling Alice Barkan lab LingYa Liao