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J. Biol. Chem., Vol. 279, Issue 40, 41495-41503, October 1, 2004

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Regulation of the Fusion Pore Conductance during Exocytosis by Cyclin-dependent Kinase 5^*

Jeff W. Barclay, Marcos Aldea, Tim J. Craig¶, Alan Morgan, and Robert D. Burgoyne||

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

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.

Received for publication, June 15, 2004 , and in revised form, July 23, 2004.

^* This work was supported by grants from the Wellcome Trust and the Biotechnology and Biological Science Research Council (to R. D. B. and A. M.). 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 postdoctoral fellowship from the Natural Science and Engineering Research Council of Canada.

A fellow of the Ministerio de Ciencia y Tecnología. Present address: Departamento de Farmacología, Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.

^¶ 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}liverpool.ac.uk.

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