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First published online 3 February 2004
doi: 10.1242/jcs.00941

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Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

Department of Pharmacology, University of Vienna, Waehringerstrasse 13a, 1090 Vienna, Austria

^* Author for correspondence (e-mail: helmut.kubista{at}univie.ac.at)

Accepted 15 October 2003

The core complex, formed by the SNARE proteins synaptobrevin 2, syntaxin 1 and SNAP-25, is an important component of the synaptic fusion machinery and shows remarkable in vitro stability, as exemplified by its SDS-resistance. In western blots, antibodies against one of these SNARE proteins reveal the existence of not only an SDS-resistant ternary complex but also as many as five bands between 60 and >200 kDa. Structural conformation as well as possible functions of these various complexes remained elusive.

In western blots of protein extracts from PC12 cell membranes, an antibody against SNAP-25 detected two heat-sensitive SDS-resistant bands with apparent molecular weights of 100 and 230 kDa. A syntaxin antibody recognized only the 230 kDa band and required heat-treatment of the blotting membrane to detect the 100 kDa band. Various antibodies against synaptobrevin failed to detect SNARE complexes in conventional western blots and detected either the 100 kDa band or the 230 kDa band on heat-treated blotting membranes.

When PC12 cells were exposed to various extracellular K⁺-concentrations (to evoke depolarization-induced Ca²⁺ influx) or permeabilized in the presence of basal or elevated free Ca²⁺, levels of these SNARE complexes were altered differentially: moderate Ca²⁺ rises (1 µM) caused an increase, whereas Ca²⁺ elevations of more than 1 µM led to a decrease in the 230 kDa band. Under both conditions the 100 kDa band was either increased or remained unchanged.

Our data show that various SDS-resistant complexes occur in living cells and indicate that they represent SNARE complexes with different structures and diverging functions. The distinct behavior of these complexes under release-promoting conditions indicates that these SNARE structures have different roles in exocytosis.

Key words: SNARE, PC12, Transmitter release, SNAP-25, Syntaxin, Synaptobrevin

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