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The protein complexin and its role in coupling secretory vesicles and Ca2+
channelsQuintana J., Rohan J., Chow R.H.
Department of Physiology & Biophysics and Zilkha Neurogenetic Institute,
Keck School of Medicine, University of Southern, California, Los Angeles
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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
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Background
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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
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SNARE Complex
It assist exocytosis through “zippering”
cartoon
more secretion when presentComplexin
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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
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Question
Does the protein complexin help vesicles to associate with calcium
channels?
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HypothesisCplx 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
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Methods
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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
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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
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short depolarization protocol
long depolarization protocol
Depolarization
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How to measure the IRP & RRP
∑∆C short
∑∆C long
~IRP
~RRP
Adapted from Voets et al., Neuron 23, 607 (1999).
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Capacitance Data
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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.
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Acknowledgements
• Dr. Robert Chow• Chow’s Lab
– Joyce G. Rohan– Madison Zitting– Alan Okada
• Dr. Emil Bogenmann• NIH
– Step Up Program
ZNI
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Question