scholarly journals Synaptobrevin-2 C-terminal Flexible Region Regulates the Discharge of Catecholamine through the Fusion Pore

2018 ◽  
Author(s):  
Annita N. Weiss
Keyword(s):  
Membrane Potential ◽  
Lipid Bilayer ◽  
Chromaffin Cells ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Flash Photolysis ◽  
Fusion Pore ◽  
Snare Protein ◽  
Head Groups ◽  
Flexible Region

AbstractThe discharge of neurotransmitters from vesicles is a regulated process. Synaptobrevin-2 a SNARE protein, participates in this process through its interaction with other SNARE and associate proteins. Synaptobrevin-2 transmembrane domain is embedded into the vesicle lipid bilayer except for its last three residues. These residues are hydrophilic and constitute synaptobrevin-2 C-terminal flexible region. This region interacts with the intravesicular lipid bilayer phosphate head groups to initiate the fusion pore formation. Here it is shown that, this region also modulates the intravesicular membrane potential thereby the discharged of catecholamine. Synapotobrevin-2 Y113 residue was mutated to lysine or glutamate. The effects of these mutations on the exocytotic process in chromaffin cells were assessed using capacitance measurements, combined with amperometry and stimulation by flash photolysis of caged Ca2+. Both Y113E and Y113K mutations reduced the amplitudes of vesicle fusions and reduced the rates of release of catecholamine molecules in quanta release events. Further investigation revealed that the proximity of these charged residues near the vesicle lipid bilayer most likely changed the intravesicular potential, thereby slowing the flux of ions through the fusion pore, hence reducing the rate of catecholamine secretion. These results suggest that catecholamine efflux is couple with the intravesicular membrane potential.

scholarly journals Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

1997 ◽  
Vol 136 (5) ◽  
pp. 995-1005 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sofya A. Brener ◽  
Dong C. Ok ◽  
Fredric S. Cohen
Keyword(s):  
Outer Membrane ◽  
Negative Curvature ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Positive Curvature ◽  
Fusion Pore ◽  
Spontaneous Curvature ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Fusion Pores

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

scholarly journals The Lipid-anchored Ectodomain of Influenza Virus Hemagglutinin (GPI-HA) Is Capable of Inducing Nonenlarging Fusion Pores

2000 ◽  
Vol 11 (4) ◽  
pp. 1143-1152 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Fredric S. Cohen ◽  
Grigory B. Melikyan
Keyword(s):  
Influenza Virus ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Fusion Pore ◽  
Influenza Virus Hemagglutinin ◽  
Initial Membrane ◽  
Optimal Fusion ◽  
Pass Through

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.

scholarly journals Nanometer resolution in situ structure of SARS-CoV-2 post-fusion spike

2021 ◽  
Author(s):  
Yun Zhu ◽  
Fei Sun ◽  
Xiangxi Wang ◽  
Linhua Tai ◽  
Guoliang Zhu ◽  
...  
Keyword(s):  
Pore Formation ◽  
Transmembrane Domain ◽  
Structural Information ◽  
Protein S ◽  
Fusion Pore ◽  
Electron Microscopic ◽  
Nanometer Resolution ◽  
Entry Mechanism ◽  
Resolution Structure

Abstract The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of viral genome into host cell. To understand its detailed entry mechanism and develop specific entry inhibitor, the in situ structural information of SARS-CoV-2 spikes in different states are urgently important. Here, by using the cryo-electron microscopic tomograms, we observed spikes of inactivated SARS-CoV-2 virions in both pre-fusion and post-fusion states and solved the nanometer resolution structure of in situ post-fusion spike. With a more complete model compared to previous reports, the relative spatial position between fusion peptide and transmembrane domain was discovered. Novel oligomerizations of spikes on viral membrane were observed, likely suggesting a new mechanism of fusion pore formation.

scholarly journals Fatty Acids on the A/USSR/77 Influenza Virus Hemagglutinin Facilitate the Transition from Hemifusion to Fusion Pore Formation

2002 ◽  
Vol 76 (9) ◽  
pp. 4603-4611 ◽  
Author(s):  
Tatsuya Sakai ◽  
Reiko Ohuchi ◽  
Masanobu Ohuchi
Keyword(s):  
Influenza Virus ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Fusion Reaction ◽  
Biological Significance ◽  
Site Directed Mutagenesis ◽  
Fusion Pore ◽  
Influenza Virus Hemagglutinin ◽  
Calcein Leakage ◽  
Palmitic Acids

ABSTRACT Influenza virus hemagglutinin (HA) has three highly conserved acylation sites close to the carboxyl terminus of the HA2 subunit, one in the transmembrane domain and two in the cytoplasmic domain. Each site is modified by palmitic acid through a thioester linkage to cysteine. To elucidate the biological significance of HA acylation, the acylation sites of HA of influenza virus strain A/USSR/77 (H1N1) were changed by site-directed mutagenesis, and the membrane fusion activity of mutant HAs lacking the acylation site(s) was examined quantitatively using transfer assays of lipid (R18) and aqueous (calcein) dyes. Lipid mixing, so-called hemifusion, activity was not affected by deacylation, whereas transfer of aqueous dye, so-called fusion pore formation, was dramatically restricted. When the fusion reaction was induced by a lower pH than the optimal one, calcein transfer with the mutant HAs was improved, but simultaneously a considerable calcein leakage into the medium was observed. From these results, we conclude that the palmitic acids on the H1 subtype HA facilitate the transition from hemifusion to fusion pore formation.

Mechanistic Insights into Pore Formation by an α-Pore Forming Toxin: Protein and Lipid Bilayer Interactions of Cytolysin A

2020 ◽  
Vol 54 (1) ◽  
pp. 120-131
Author(s):  
Pradeep Sathyanarayana ◽  
Sandhya S. Visweswariah ◽  
K. Ganapathy Ayappa
Keyword(s):  
Lipid Bilayer ◽  
Pore Formation ◽  
Toxin Protein

scholarly journals Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

2018 ◽  
Vol 151 (2) ◽  
pp. 118-130 ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Small Molecules ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Chromogranin B ◽  
Tissue Plasminogen ◽  
Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting <1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

CAN SINGLE ELECTRONS INITIATE FUSION OF BIOLOGICAL MEMBRANES?

2007 ◽  
Vol 02 (01) ◽  
pp. 23-31 ◽  
Author(s):  
MARTIN KOCH ◽  
ANGELA M. OTTO ◽  
JOACHIM WIEST ◽  
BERNHARD WOLF
Keyword(s):  
Conformational Changes ◽  
Pore Formation ◽  
Aromatic Amino Acids ◽  
Fusion Pore ◽  
Mathematical Approach ◽  
Animal Cells ◽  
Lipid Complex ◽  
Single Electrons ◽  
Initial Event ◽  
Membrane Barrier

Animal cells export the content of vesicles by exocytosis, a process in which a vesicle and the plasma membrane fuse and ultimately form a pore. The initial event leading to the breakthrough of the membranes for pore formation is not well understood. Here we present a mathematical approach which suggests that this process is initiated by single electrons which could be present in the immediate proximity of the fusing membranes in aromatic amino acids. The electron can be regarded as non-classical; it takes up energy and transfers it to the membrane barrier, thereby eliciting conformational changes in the proteo-lipid complex leading to membrane fusion and thus initiating the opening of the fusion pore.

scholarly journals The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment

2019 ◽  
Vol 1861 (3) ◽  
pp. 670-676 ◽  
Author(s):  
Zahia Fezoua-Boubegtiten ◽  
Benoit Hastoy ◽  
Pier Scotti ◽  
Alexandra Milochau ◽  
Katell Bathany ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Snare Protein ◽  
Highly Sensitive ◽  
Lipid Environment
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