[Abstract Title]. - Society for Neuroscience

Title: Neurosecretion mediated by hetero-oligomeric SNARE supercomplexes in chromaffin
cells
Authors: *R. MOHRMANN, J. B. SORENSEN;
Dept of Membrane Biophys, Max-Planck Inst. f Biophys Chem, Göttingen, Germany
Abstract: Vesicle fusion requires the orchestrated interaction of multiple key components of the
release machinery, including the formation of SNARE-complexes. It is envisioned that several
SNARE complexes accumulate at the prospective fusion site to form a ring-like supercomplex,
in the middle of which the fusion pore forms. Here, we present a strategy to gain insight into
supercomplex structure and function by progressive inhibition of fusion using a dominant
negative SNAP25 variant. This SNAP25 version bears a double point mutation in layer +5, does
not allow rescue when expressed in Snap-25 null cell, and abolish fusion when overexpressed in
wildtype cells. The dependency of inhibition on the concentration of the dominant-negative
variant can deliver information on the properties of supercomplexes. In order to measure the
inhibition profile, wildtype SNAP25 and dominant-negative variant were co-expressed in Snap-
25 null chromaffin cells using dicistronic Semliki-viruses harboring an internal ribosomal entry
site (IRES) of either polio- or encephalomyocarditis-virus. To obtain different expression ratios,
viruses with different succession of the reading frames of both SNAP25 versions and different
IRES-elements were generated. Both SNAP25 versions were N-terminally tagged with GFP or
mCherry and the expression ratio determined by fluorescence quantification. N-terminal tagging
did not affect the function of the protein. Secretion was assayed by patch-clamp capacitance
measurements and amperometry and elicited by flash photolysis of caged-calcium. Increasing
concentrations of the inhibitory variant decreased the amount of exocytosis. The inhibition
profile displayed a super-linear dependence upon the concentration of the inhibitory variant,
indicating the existence of supercomplexes, i.e. fusion complexes containing more than one copy
of SNAP-25. Inhibition affected the fast release component more dramatically than overall
release, suggesting that ready-releasable vesicles depend on supercomplexes with a larger
number of SNAREs - or with a more defined configuration of these complexes - than the
sustained release component. This analysis shed new light on the dependence of exocytosis on
the SNARE-complex, and delivers an explanation of the wide-spread observation that the fast
release component is more dramatically affected than sustained released by genetic manipulation
of exocytotic proteins.
Disclosures: R. Mohrmann, None; J.B. Sorensen, None.
Poster
236. Neurotransmitter Release: Docking and Fusion
Time: Sunday, November 16, 2008, 1:00 pm - 5:00 pm
Program#/Poster#: 236.17/D17