Time-Resolved Dynamics of the Vesicle Membrane During Individual Exocytotic Secretion Events, as Extracted from Amperometric Monitoring of Adrenaline Exocytosis from Chromaffin Cells

Abstract

In chromaffin cells, adrenaline is known to be released through docking and then fusion of a secretory vesicle to the cytoplasmic membrane of the cell. Here we propose a method for the calculation of the dynamics of the vesicle membrane during the fusion from amperometric currents observed during individual exocytotic secretion events. The method is based on recognition of the fact that the overall current spike shape results from the convolution of the membrane dynamics with the rate of diffusion and exchange of the catecholamine cation inside the matrix core of the vesicle. This convolution can be treated analytically thanks to a reasonable approximation on the relative time scales of the opening function and diffusion; this leads to a convolution integral with which one deconvolutes the experimental amperometric data. An alternative numerical treatment through Brownian motion simulations dispenses with the need for this simplifying approximation. Combination of both approaches yields the membrane dynamics with a precision and a time resolution never achieved before. The peculiar dynamics of the vesicle membrane hint that exocytotic events are regulated by the swelling of the matrix polyelectrolyte core of the vesicle (although this important component is transparent in the analysis proposed here); this points to the important role of matrix swelling in exocytotic behavior. In particular, the effect may be elaborated to offer a new energetic interpretation of the transition between pore release and fusion release: secretory vesicles which involve pores and matrices similar to those of the adrenal cells investigated here can be separated into two classes according to their radius and catecholamine content. Small vesicles (`ca. 25 nm radius, and containing ca. 20 000 molecules) should always release their contents through pore docking; larger vesicles should always fuse, unless another mechanism closes the pore before ca. 20 000 molecules of catecholamine have been released.

‘Full fusion’ is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

“…From a mechanical perspective, both changes are in agreement with the expected decrease or increase, respectively, of the surface tension, i.e. of the driving force powering the fusion pore expansion [34][35][36][75][76][77]. Nonetheless, one observes that R max pore < 30 nm in all cases (normal, hypertonic or hypotonic conditions), suggesting that this limit does not depend on the membrane characteristics, although, as expected, it is more frequently reached for tense membranes than for relaxed ones.…”

“…Still, it is noted that such a value is not incompatible with the mesh sizes of sub-membrane cytoskeleton proteic structures [88][89][90][91]. Before the fusion pore external edge meets this limit, its radial expansion rate results from a balance between the driving force acting on the edge of the pore and the ability of the system to relax its released energy through viscous dissipation [35,36,75,76]. Accordingly, R pore (t) enlarges exponentially at the beginning of its expansion (i.e.…”

“…If the fusion pore dynamics is exclusively governed by bilipid membrane energetics [34][35][36], its enlargement should be driven by the viscous dissipation of tension energies imposed by the important curvatures created at the small fusion pore edges and by the vesicle membrane surface tension [32,[34][35][36]. The replacement of catecholamine cations by hydrated monovalent ones inside the matrix during release should lead to swelling of the pore [37][38][39][40].…”

“…However, swelling is necessarily restrained by the presence of the vesicle membrane, at least while the pore radius remains small compared with that of the vesicle [32]. The ensuing internal swelling pressure that builds up in the constrained matrix during release provokes a constant increase in the membrane surface tension and sustains the continuous enlargement of the fusion pore [35,36]. So, in the absence of external factors, 'full fusion' appears ineluctable.…”

Fabrication of a NCD microelectrode array for amperometric detection with micrometer spatial resolution

“…The current density j obtained from Eq. (1) for the oxidation of adrenaline is approximately 40 μA/cm 2 , assuming D~6 ·10 6 cm 2 /s [35,36] and r 0 = 4 μm. This value fits reasonably our data, considering that the geometry of microelectrodes is not hemi-spherical and that a precise evaluation of the active area should consider the nanocrystalline morphology of the NCD surface.…”