Temporally resolved, independent stages of individual exocytotic secretion events

Schroeder, Timothy; Borges, Ricardo; Finnegan, Jennifer M.; Pihel, Karin; Amatore, Christian; Wightman, R. Mark

doi:10.1016/s0006-3495(96)79652-2

Cited by 142 publications

(162 citation statements)

References 46 publications

(54 reference statements)

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“…When no foot is observed, the Gaussian distribution of event area exhibits a shoulder toward smaller amounts of transmitter released. In agreement with previous work (Schroeder et al, 1996), however, there is not a significant shift in the distribution. A similar histogram for data obtained from L-DOPA-treated PC12 cells is shown in Figure 3B.…”

Section: Foot Frequency As a Function Of Vesicular Contentsupporting

confidence: 93%

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

Sombers¹,

et al. 2004

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Many spikes in amperometric records of exocytosis events initially exhibit a prespike feature, or foot, which represents a steady-state flux of neurotransmitter through a stable fusion pore spanning both the vesicle and plasma membranes and connecting the vesicle lumen to the extracellular fluid. Here, we present the first evidence indicating that vesicular volume before secretion is strongly correlated with the characteristics of amperometric foot events. L-3,4-Dihydroxyphenylalanine and reserpine have been used to increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles. Amperometry and transmission electron microscopy have been used to determine that as vesicle size is decreased the frequency with which foot events are observed increases, the amount and duration of neurotransmitter released in the foot portion of the event decreases, and vesicles release a greater percentage of their total contents in the foot portion of the event. This previously unidentified correlation provides new insight into how vesicle volume can modulate the activity of the exocytotic fusion pore.

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Section: Foot Frequency As a Function Of Vesicular Contentsupporting

confidence: 93%

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

Sombers¹,

et al. 2004

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“…ATP in chromaffin granules has a structure similar to crystalline ATP, not to ATP in solution 23 , suggesting a low free ATP concentration. The fusion pore models indicate intravesicular free catecholamine concentrations of 130-370 mM, much lower than the total vesicular catecholamine concentration 24 , which is consistent with binding to the granular matrix 25,26 or alternatively catecholamines and ATP forming a highly non-ideal solution 27 . Experiments employing solutions where Cl − ions are substituted by large anions, with much lower diffusion coefficients or even too large to permeate the fusion pore should allow a better estimate of the fusion pore permeability for anions.…”

Section: Discussionmentioning

confidence: 59%

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

2007

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Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. We tested this hypothesis in chromaffin cells using cell-attached patch amperometry measuring simultaneously catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% (if any) of cations enter the vesicle through its membrane. Instead we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na + influx through the fusion pore as predicted by electrodiffusion theory applied to fusion pore permeation and suggest a prefusion rather than postfusion role for vesicular cation channels.Neurotransmitter and hormone release occurs by exocytosis, which begins with the formation of a narrow fusion pore 1,2 . Fusion pores in mast cells and chromaffin cells have typically an initial conductance of ~330 pS 1,3 through which vesicular serotonin and catecholamines are released, respectively 4,5 . The initial fusion pore of a synaptic vesicle is typically >280 pS and release of neurotransmitter should occur with a time constant <500 μs due to the small vesicle volume 6 . Many neurotransmitters and hormones are organic ions that carry charge with them. Among these acetylcholine, serotonin and catecholamines are mostly monovalent cations at physiological or more acidic vesicular pH. Efflux of charged molecules through a narrow fusion pore would rapidly charge the small capacitance of the vesicle and a mechanism of charge compensation is required.Release of catecholamines from a single vesicle can be detected electrochemically 7 . The initial flux of catecholamines through the early narrow fusion pore can be measured as an amperometric foot signal preceding the amperometric spike 3,5,8 . The amplitude of amperometric foot currents is typically ~5 pA. Since most catecholamine molecules will be in monovalent cationic form but two electrons are transferred per molecule in the oxidation giving rise to the amperometric current 9 , the catecholamines released during the foot signal carry an ionic current of ~2.5 pA with them. This would charge a typical bovine chromaffin granule 4correspondence should be addressed to M.L. (ml95@cornell.edu).

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“…All peaks identified by the program were inspected visually, and overlapping peaks were excluded manually from the data sets. Additionally, if a spike had a zero correlation with an exponentially modified gaussian f unction, it also was excluded (Jankowski et al, 1994;Schroeder et al, 1996). On average, this was ϳ5% of the well resolved spikes.…”

Section: Methodsmentioning

confidence: 99%

VMAT-Mediated Changes in Quantal Size and Vesicular Volume

et al. 2000

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It has been well established that the volume of secretory vesicles can be modulated. However, we present the first data demonstrating that the amount of transmitter in a vesicle can regulate its volume. Amperometry and transmission electron microscopy have been used to determine that L-3,4-dihydroxyphenylalanine and reserpine increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles as well as their catecholamine content. Because changes in vesicular catecholamine content are tracked by changes in vesicle volume, our results indicate that when quantal size is altered via the vesicular monoamine transporter the concentration of catecholamines within the vesicles remains relatively constant. This previously unidentified cellular response provides new insight into how catecholamines can be packaged in and released from secretory vesicles.

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Temporally resolved, independent stages of individual exocytotic secretion events

Cited by 142 publications

References 46 publications

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

VMAT-Mediated Changes in Quantal Size and Vesicular Volume

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