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

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

Background

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

SNARE Complex

It assist exocytosis through “zippering”

cartoon

more secretion when presentComplexin

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

Question

Does the protein complexin help vesicles to associate with calcium

channels?

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

Methods

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

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

short depolarization protocol

long depolarization protocol

Depolarization

How to measure the IRP & RRP

∑∆C short

∑∆C long

~IRP

~RRP

Adapted from Voets et al., Neuron 23, 607 (1999).

Capacitance Data

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.

Acknowledgements

• Dr. Robert Chow• Chow’s Lab

– Joyce G. Rohan– Madison Zitting– Alan Okada

• Dr. Emil Bogenmann• NIH

– Step Up Program

ZNI

Question