(Invited) Amperometry Detection of Full Fusion Mode of Catecholamine Exocytosis from Single PC12 Cell Elicited By Sodium Dodecyl Sulfate
Authors: Hu Ren , Oleinick Alexander , Svir Irina , Ren Bin , Lin Chang-Jian , Zhan Dong-Ping , Amatore Christian Andre , Tian Zhong-Qun 
Published: February 2020

Vesicular exocytosis is a crucial process for living cells by which signalling species such as acetylcholine and catecholamines and other vesicular contents can be secreted into the extracellular environment. Single cell electrochemistry, especially the “semi-artificial synapse” based amperometry, has been used to scrutinize exocytosis dynamics of single vesicles due to its high sensitivity and appropriate temporal resolution. Important characteristics of vesicular release as well as the effects of biological, chemical and physical parameters on them have thus been evaluated for many cell lines. This has led to contest the conventional full release mode. Recently, the view that partial release is the most common for most neurotransmitters stored in dense core vesicles has gained acceptance. However, the fundamental reasons leading to partial fusion remain an open question waiting experimental characterization.

In this work, catecholamine release was elicited from PC12 using sufficiently small concentrations of sodium dodecyl sulfate (SDS) to perturb normal release mechanism in an attempt to reveal concealed information while keeping physiologically compatible conditions. Amperometry was used to monitor and quantify the released fluxes and kinetics of individual vesicular events based on analyses of statistically significant series of events. This evidenced that stimulating release with SDS 350 μM leads to a doubling of the quantity of catecholamine cations released per event and much larger release fluxes compared to controls (release elicited with K+105 mM under same conditions) or SDS 250 μM. Importantly, the releases stimulated by SDS 350 μM showed strict dependence on calcium ion, indicating the biological significance of the release.

These quantitative measurements provide substantial proof for the partial release hypothesis. The results confirm our previous theoretical model and are consistent with ex situ cytometric experiments on isolated PC12 vesicles reported in the literature, which established that release is far from being total under normal exocytotic conditions. Based on the theoretical model, these data established that the maximal size of fusion pores at the end of the “full fusion” phase is limited by some constriction of the fusion pore radius. Indeed, the present results are entirely consistent with the fact that SDS 350 μM allows the fusion pore to expand to a double size (ca. 28 nm radius) compared to controls and SDS 250 μM (ca. 14 nm radius).

Acknowledgements:

We thank the MOST of China (2016YFC1100300 and 2013CB933703), the NSFC (51571169 and 21303146) and the Fundamental Research Funds for the Central Universities (20720170031) for financial support of the work performed in Xiamen. CNRS, ENS and UPMC (UMR 8640) supports are acknowledged for that performed in Paris. We also thank our joint laboratory, LIA CNRS NanoBioCatEchem, for supporting the collaboration between the two research teams, as well as discussions with Jerome Delacotte (ENS, UMR 8640).