[Abstract Title]. - Society for Neuroscience

Title: Phosphatidylserine regulates fusion pores in PC12 cells
Authors: *Z. ZHANG 1 , E. HUI 2 , E. R. CHAPMAN 1 , M. B. JACKSON 1 ;
1 Dept Physiol, UW-Madison, Madison, WI; 2 Biophysic program, Univ. of Wisconsin, Madison,
Madison, WI
Abstract: Synaptotagmin (syt) I senses Ca 2+ and triggers exocytosis by initiating a sequence of
molecular interactions. Syt I binds to both SNARE proteins and lipid bilayers containing
phosphatidylserine (PS) in a Ca 2+ -dependent manner, but the precise functions of these
interactions in exocytosis have been difficult to establish. The present study investigated the role
of PS binding by using carbon fiber amperometry to record Ca 2+ -triggered norepinephrine
release from PC12 cells. The PS content of the inner leaflet of the plasma membrane was altered
either by lipid addition to the growth medium or by overexpression of PS synthase 1, PS
synthase 2, or their function-enhancing mutants. The PS content of PC12 cells was then
determined by HPLC. All manipulations that elevated PS levels in PC12 cells enhanced
exocytosis by increasing the frequency of vesicle fusion events triggered by Ca 2+ . Elevated PS
levels also increased the lifetime of fusion pores that opened during release events. At the highest
PS level attained, the average fusion pore duration was more than double that of controls.
Addition of phosphatidylcholine had no effect on exocytosis or fusion pores. Analysis of fusion
pore openings that close (kiss-and-run), together with fusion pore openings that dilate (fullfusion),
indicated that PS had a weak, insignificant effect on the rate of fusion pore closure but
strongly reduced the rate of fusion pore dilation. Elevating PS levels failed to alter vesicle size or
voltage-activated calcium current. Staurosporine produced very small reductions in the effects of
PS on fusion pore lifetime, indicating that the major component of PS action does not depend on
protein kinase C. Stopped-flow kinetics measurements of Ca 2+ -stimulated binding of syt to PS
containing liposomes showed that the binding became stronger as PS content was increased.
Thus, the effects of PS revealed here may indicate roles for syt-lipid interactions in exocytosis
and fusion pore stability.
Disclosures: Z. Zhang, None; E. Hui, None; E.R. Chapman, None; M.B. Jackson, None.
Poster
236. Neurotransmitter Release: Docking and Fusion
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 236.5/D5
Topic: B.06.a. Docking and fusion
Support: Slovenian Research Agency Grant P3 310