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J. Biol. Chem., Vol. 280, Issue 36, 31615-31623, September 9, 2005

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Amisyn Regulates Exocytosis and Fusion Pore Stability by Both Syntaxin-dependent and Syntaxin-independent Mechanisms^*

John R. L. Constable, Margaret E. Graham, Alan Morgan, and Robert D. Burgoyne

From the Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, United Kingdom

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca²⁺-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.

Received for publication, May 31, 2005 , and in revised form, July 20, 2005.

^* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Wellcome Trust Prize Studentship.

To whom correspondence should be addressed. Tel.: 44-151-794-5305; Fax: 44-151-794-5337; E-mail: burgoyne{at}liv.ac.uk.

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