Three Distinct Modes of Exocytosis Revealed by Amperometry in Neuroendocrine Cells

Abstract

Neurotransmission requires Ca(2+)-dependent release of secretory products through fusion pores that open and reclose (partial membrane distention) or open irreversibly (complete membrane distention). It has been challenging to distinguish between these release modes; however, in the work presented here, we were able to deduce different modes of depolarization-evoked exocytosis in neuroendocrine chromaffin and PC12 cells solely by analyzing amperometric recordings. After we determined the quantal size (Q), event half-width (t(50)), event amplitude (I(peak)), and event decay time constant (τ(decay)), we fitted scatter plots of log-transformed data with a mixture of one- and two-dimensional Gaussian distributions. Our analysis revealed three distinct and differently shaped clusters of secretory events, likely corresponding to different modes of exocytosis. Complete membrane distention, through fusion pores of widely varying conductances, accounted for 70% of the total amount of released catecholamine. Two different kinds of partial membrane distention (kiss-and-run and kiss-and-stay exocytosis), characterized by mode-specific fusion pores with unitary conductances, accounted for 20% and 10%, respectively. These results show that our novel one- and two-dimensional analysis of amperometric data reveals new release properties and enables one to distinguish at least three different modes of exocytosis solely by analyzing amperometric recordings.

Identification of a New Exo-Endocytic Mechanism Triggered by Corticotropin-Releasing Hormone in Mast Cells

“…However, the augmentation of the granular content could be a result of compound fusion (i.e., two or more SG that fuse before exocytosis) (50). Previous analysis of amperometric spikes has indicated that different modes of exocytosis could be reflected in several characteristics, such as the spike half-width or spike rise (39,51,52). Moreover, a previous study suggested that amperometric feet correspond to SG with higher content (35), similar to what we observed in our study (Table II).…”

“…Following cell stimulation, a brief current in the form of spikes can be observed as a consequence of the oxidation of the transmitters released by SG (31,38). This technique allows for the detection of differences in the amount of product secreted per SG and cell (39). Therefore, we studied the effects of CRH and Ag (DNP) on the characteristics of exocytotic events.…”

Amperometric Measurements at Cells Support a Role for Dynamin in the Dilation of the Fusion Pore during Exocytosis

“…The distribution of the exocytotic parameters is asymmetric and strongly deviates from normality, hence, motivating the use of the median in place of the mean. [14],[17],[28],[29] These data reveal that inhibition of dynamin GTPase activity leads to significant decreases in the characteristic peak times, t 1/2 and t fall, in the peak current i p and in the released number of molecules, N . Interestingly, this effect is more important when the concentration of dynasore increases.…”

Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands

“…Figure 4A reports the scatter plot of Log(Q(fC)) versus Log(I max (pA)) values, where variances are represented by solid curves. Three 2D Gaussian distributions uncovered the existence of different clusters of large, small and SAF events, in analogy with 35 . For all the events we found direct proportionality between Log(Q(fC)) and…”

“…These features make the chip suitable for addressing relevant physiological issues related to the detection of spontaneous and stimulated catecholamine secretion, as well as the discrimination between events associated with complete ("full fusion") and incomplete vesicle fusion ("kiss-and-run" and "kiss-and-stay" exocytosis) 35 . In particular, the relevance of spontaneous and evoked secretion is related to the in vivo supply of catecholamines from the adrenal gland that has to be continuously tuned on the basis of the hormonal need, thus ranging from a basal resting release to a more sustained secretion triggered by continuous cell stimulation mimicking stressful conditions.…”