Compl

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Compl

  1. 1. The protein complexin and its role in coupling secretory vesicles and Ca2+ channels Quintana J., Rohan J., Chow R.H. Department of Physiology & Biophysics and Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern, California, Los Angeles
  2. 2. Big Picture • understand better how secretion is regulated • use the acquired knowledge to create a solution against diseases which there is not enough secretion • Example: type 2 diabetes
  3. 3. Background
  4. 4. Hormones Exocytosis • signaling molecules • stored in vesicle • example: insulin • process by which the membrane of a vesicle fuses with the cell plasma membrane, and the vesicle contents are secreted Vesicles • to be readily releasable, they have to be docked and primed
  5. 5. SNARE Complex It assist exocytosis through “zippering” cartoon more secretion when present Complexin
  6. 6. Calcium & Ca2+ Channels • triggers exocytosis • vesicles nearer to Ca2+ channels secrete more reliably • Readily Releasable Pool (RRP): primed vesicles both near and far from Ca2+ channels • Immediately Releasable Pool (IRP): primed vesicles near Ca2+ channels RRP & IRP
  7. 7. Question Does the protein complexin help vesicles to associate with calcium channels?
  8. 8. Hypothesis Cplx facilitates co-localization of vesicles and Ca2+ channels. We expect to see a change in the IRP if a cell is missing Cplx (i.e. Cplx knock out) IRP (WT) IRP (KO) > RRP RRP Wild Type Knock Out RRP IRP RRP IRP
  9. 9. Methods
  10. 10. Genotyping • KO mice – Adrenal chromaffin cells from litter mates of Cplx +/-(hemizygous) mice KO Bands WT Bands L 1 2 3 4 5 6 7 8 9 10 11 HZ WT KO
  11. 11. Patch Clamp • biophysical tool • Capacitance (C): electrical measurement that is proportional to the area of the cell membrane • IRP = ∑∆C short • RRP = ∑∆C short + ∑∆C long Modified from Sakmann and Neher, Sci. Am. 1991
  12. 12. short depolarization protocol long depolarization protocol Depolarization
  13. 13. How to measure the IRP & RRP ∑∆C short ∑∆C long ~IRP ~RRP Adapted from Voets et al., Neuron 23, 607 (1999).
  14. 14. Capacitance Data
  15. 15. Conclusion • Our preliminary data supports our hypothesis that Cplx helps co-localize vesicles and Ca2+ channels. • Data needs to be replicated and statistically validated. • If validated, then Cplx serves not only to help prime vesicles, but also to increase reliability of stimulus-secretion coupling.
  16. 16. Acknowledgements • Dr. Robert Chow • Chow’s Lab – Joyce G. Rohan – Madison Zitting – Alan Okada • Dr. Emil Bogenmann • NIH – Step Up Program ZNI
  17. 17. Question

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