Molecular composition and regulation of the fusion pore of calcium triggered exocytosis.

摘要

Ca2+-triggered neurotransmitter release is critical for synaptic transmission. It starts with an intermediate structure, known as the fusion pore. In the present study, amperometry and capacitance measurements were used to examine the fusion pore of large dense core vesicles in neuroendocrine PC12 cells. Genetic manipulation of the fusion machinery in PC12 cells was carried out by overexpressing the proteins of interest separately from a green fluorescent protein reporter. Regulation of the fusion pore was interpreted by applying single channel kinetic analysis.;In this work, the fusion pore flux was shown to be sensitive to manipulations of the side chain size or charge of certain residues in the syntaxin (syx) membrane anchor. These residues fell on the same face of the syx membrane alpha-helix, and thus were predicted to line the fusion pore. Based on the fusion pore conductance, 5--8 copies of the syx membrane alpha-helix in a circular arrangement were estimated to form the fusion pore in the plasma membrane.;Fusion pores open and soon dilate with a mean life time of ∼2 msec. Manipulations of the syx membrane anchor altered the life time of an open fusion pore. Based on a simple kinetic scheme that an open fusion pore can either dilate or close, one can estimate changes in the kinetic parameters of a fusion pore. Syx pore lining residues appear to regulate the fusion pore dilation rate. This result is consistent with the notion that these residues would experience a transition during dilation, from a hydrophilic environment to a hydrophobic environment.;The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex has been proposed to serve an essential role in exocytosis. In the present study, mutations that reduced the SNARE complex thermostability were shown to slow the rate of secretion and the rate of fusion pore dilation. A fully formed SNARE complex was demonstrated to be important to complete exocytosis. However, only part of the SNARE complex experiences a conformational transition to dilate an open fusion pore.