The fusion pore

Lindau, Manfred; Toledo, G. Álvarez de

doi:10.1016/s0167-4889(03)00085-5

Cited by 151 publications

(154 citation statements)

References 67 publications

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“…Additionally, previous work using single-cell carbon fiber amperometry [30,31] has indicated that quantal size positively depends on cell Ca 2+ levels [32] by transitioning from Ω-form fusion transients to 'full collapse' events, thus expelling all granule contents [3,8,9]. We measured the quantal size of catecholamine secretion in Dyn transfected cells compared to untransfected controls to determine the relative occurrence of Ω-form 'kiss an run' to 'full collapse'.…”

Section: A Role For Dynamin I In High Frequency-evoked Exocytosismentioning

confidence: 99%

“…Stabilization of the pore before dilation also acts to maintain segregation of the granule and cell plasma membrane [7]. Reversal of the fusion pore represents the endocytosis of the granule membrane, terminating the fusion event prior to release of the dense granule core [3,8,9]. The molecular mechanism of fusion pore regulation has received much attention over the years.…”

Section: Introductionmentioning

confidence: 99%

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Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Fulop

Doreian

Smith

2008

Archives of Biochemistry and Biophysics

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Under low stimulation, adrenal chromaffin cells release freely-soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.

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Section: A Role For Dynamin I In High Frequency-evoked Exocytosismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Fulop

Doreian

Smith

2008

Archives of Biochemistry and Biophysics

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“…For solution A the conductivity of the solution filling the fusion pore is approximately σ=15 mScm −1 . The fusion pore conductance G P can be written as (2) and we can substitute the pore geometry in eq. 1 giving…”

Section: Electrodiffusion Calculationsmentioning

confidence: 99%

“…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 .…”

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confidence: 99%

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

2007

Self Cite

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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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“…Vesicle fusion proceeds through membrane intermediates of stalk formation, fusion pore opening, and fusion pore expansion (Lindau and Alvarez de Toledo, 2003). In classical modes of vesicle exocytosis, fusion pores expand to the point where the vesicle membrane flattens on the plasma membrane (full fusion), leading to complete luminal contents release (full release).…”

Section: Introductionmentioning

confidence: 99%

Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

Lynch

Gerona

Kielar

et al. 2008

MBoC

118

147

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In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca 2؉ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca 2؉ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca 2؉ -dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca 2؉ -dependent SNARE binding or in Ca 2؉ -dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of differentsized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca 2؉ -dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca 2؉ -dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca 2؉ -dependent membrane insertion and SNARE binding to drive fusion pore expansion. INTRODUCTIONNeurotransmitter and peptide hormone secretion is mediated by the Ca 2ϩ -dependent exocytosis of vesicles at the plasma membrane. Vesicle fusion proceeds through membrane intermediates of stalk formation, fusion pore opening, and fusion pore expansion (Lindau and Alvarez de Toledo, 2003). In classical modes of vesicle exocytosis, fusion pores expand to the point where the vesicle membrane flattens on the plasma membrane (full fusion), leading to complete luminal contents release (full release). However, vesicle exocytosis can utilize alternative modes in which the fusion pore either abruptly closes (kiss-and-run) or in which the fusion pore dilates but subsequently recloses (cavicapture; Henkel and Almers, 1996). These transient modes of vesicle exocytosis lead to the partial release of luminal contents depending on the size and diffusibility of the cargo (Alvarez de Toledo et al., 1993;Barg et al., 2002;Taraska et al., 2003). Ca 2ϩ levels regulate fusion pore expansion as well as fusion pore formation (Fernandez-Chacon and Alvarez de Toledo, 1995;Hartmann and Lindau, 1995;Wang et al., 2006), but the mechanisms underlying fusion pore expansion, which is the more energetically demanding step, are poorly understood (Cohen and Melikyan, 2004).A Ca 2ϩ -dependent fusion machinery mediates the triggered formation of fusion pores. Three SNARE proteins (VAMP-2 on the vesicle with SNAP25 and syntaxin-1 on the plasma membrane) form trans complexes that promote close membrane apposition and membrane fusion (Weber et al., 1998). Members of the synaptotagmin (Syt) protein family that localize to vesicles function as Ca 2ϩ sensors that couple Ca 2ϩ rises ...

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The fusion pore

Cited by 151 publications

References 67 publications

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

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

Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

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