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Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry

Artikel i vetenskaplig tidskrift
Författare Xianchan Li
J. Dunevall
L. Ren
Andrew G Ewing
Publicerad i Analytical Chemistry
Volym 89
Nummer/häfte 17
Sidor 9416-9423
ISSN 0003-2700
Publiceringsår 2017
Publicerad vid Institutionen för kemi och molekylärbiologi
Sidor 9416-9423
Språk English
Länkar 10.1021/acs.analchem.7b02226
Ämnesord adrenal chromaffin cells, dense-core vesicles, quantal size, fusion, pore, secretory vesicles, vesicular volume, single-cell, release, exocytosis, events, ow rh, 1992, nature, v356, p60
Ämneskategorier Molekylärbiologi, Kemi

Sammanfattning

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 degrees C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation.

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