KCNQ1 and Long QT Syndrome

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Presentation transcript:

KCNQ1 and Long QT Syndrome Daniel Burgardt

Long-QT Syndrome “LQTS is a disorder of cardiac repolarization resulting in QT prolongation on an electrocardiogram.”11 12

But… LQTS can lead to many other symptoms, some far more serious than a longer QT interval. Sudden loss of consciousness Seizures Sudden death

What causes these phenotypes? Many reasons, some unknown, the main reason is mutations in the KCNQ1 protein. Chromosome placement16

KCNQ1 A potassium voltage-gated channel, KQT-like subfamily, member 1 Helps shuffle potassium ions across membrane to repolarize heart cells Must be phosphorylated in order to function KCNQ1 protein structure15

Phosphorylation sites in KCNQ1? There are at least seven activating phosphorylation sites along the KCNQ1 protein15 Ion_trans KCNQ_channel 100 200 300 400 500 600 S27 S409 T513 S577 T470 S464 S468 676aa

What phosphorylates KCNQ1?

PRKAR2A/B PRKAR2A PRKAR2B But they need help attaching to target proteins.

AKAP9 Protein 14

AKAP9 as a scaffold protein The needed protein to attach the PRKAR2A/B complex to KCNQ1

Previous Experiments and the one I chose to challenge…

Why use mouse as a model organism? PRKAR2A AKAP9 PRKAR2B KCNQ1

Experiment Main Hypothesis The RII binding site in AKAP9 is important in the phosphorylation of KCNQ1. Experiment Part 1 Establish that AKAP9, PRKAR2A/B, and KCNQ1 localize to the same areas. Experiment Part 2 Test to see if changes in the AKAP9 RII binding domain effect protein binding Experiment Part 3 Screen for unknown mutations for further testing.

Do all four proteins localize to the same part of the heart cell? Method: Immunofluorescence analysis of heart cells Protein Antibody with fluorophore Localization of KCNQ1 in heart cells5

Are all four proteins active in the same cell functions? Method: Look at gene ontology terms PRKAR2A3 AKAP92 PRKAR2B4 KCNQ11

Experiment Main Hypothesis A mutation in the RII binding site in AKAP9 causes a decrease in phosphorylated KCNQ1. Experiment Part 1 Establish that AKAP9, PRKAR2A/B, and KCNQ1 localize to the same areas. Experiment Part 2 Test to see if changes in the AKAP9 RII binding domain effect protein binding Experiment Part 3 Screen for unknown mutations for further testing.

Does changes in AKAP9 RII binding sites effect the binding of PRKAR2A/B? Method: Microarray of mouse strand AKAP9 protein to look for phosphorylated PRKAR2A/B complex A: wt AKAP9 B: AKAP9 Δ RII Binding site RII Binding Domain Shows wt-AKAP9 Shows AKAP9 ΔRII Binding site ?

Experiment Main Hypothesis A mutation in the RII binding site in AKAP9 causes a decrease in phosphorylated KCNQ1. Experiment Part 1 Establish that AKAP9, PRKAR2A/B, and KCNQ1 localize to the same areas. Experiment Part 2 Test to see if changes in the AKAP9 RII binding domain effect protein binding Experiment Part 3 Screen for unknown mutations for further testing.

Is there a specific mutation in the RII binding site on AKAP9? Method: DNA sequencing of patients with Congenital Long- QT Syndrome (LQTS)

Future Directions Is the mutation that is found in the RII binding site causing a problem in localization of the PRKAR2A/B complex? Does the same interactions of proteins occur in all cells known to have these proteins? Is there a mutation in the PRKAR2A/B complex?

Questions

References 1-http://www.genecards.org/cgi-bin/carddisp.pl?gene=KCNQ1 2-http://www.genecards.org/cgi-bin/carddisp.pl?gene=AKAP9 3-http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAR2A 4-http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAR2B 5-Moretti, Alessandra et al. “Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome”. The New England Journal of Medicine. Vol. 363 No. 15. October 2010. 6-http://www.proteinatlas.org/ENSG00000127914 7-Lim, Chinten et al. “α4 Integrins are Type I cAMP-dependent protein kinase-anchoring proteins”. Nature Cell Biology. No. 9. p.415-421. 2007. 8-Barzi, Mercedes et al. “Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base”. Journal of Cell Science. No. 123. p.62-69. 2010. 9-http://www.nature.com/scitable/content/dna-sequencing-of-rt-pcr-products-can-8854580 10-Wilde, Arthur and Connie Bezzina. “Genetics of Cardiac Arrhythmias”. Heart. Vol. 91. No. 10. 2005. 11-Twedell, Diane Associate Editor. “An Overview of Congenital Long QT Syndrome”. The Journal of Continuing Education in Nursing. January/February 2005. Vol. 36, No 1. 12-http://t0.gstatic.com/images?q=tbn:ANd9GcR9yS9kVqT7GRLliNRXPf-2nv5F- 13-http://ghr.nlm.nih.gov/gene/KCNQ1 14-Protein network maps are all from: http://string.embl.de/newstring_cgi/show_input_page.pl?UserId=q9lK_ccFQ4Ys&sessionId=lDjU _IKBH5RI 15- 16- 17-