Calcium membrane fusion

Previous studies indicate that osmotic gradients promote membrane fusion, while hyperosmotic conditions inhibit membrane fusion during exocytosis. Consistent with this idea is the observation that the release of lysosomal enzymes from rabbit neutrophils, induced by the chemotactic peptide J -formylmethionyl-leucyl-phenylalanine (FMLP), is inhibited almost 80% in a 700-mosmol/kg medium. Inhibition is immediate (within 10 s), increases with osmolality, and is independent of the osmoticant. Neutrophils loaded with the calcium indicator indo-1 exhibit an FMLP-induced calcium signal that is inhibited by hyperosmolality. Hyperosmolality (700 mosmol/kg) increases basal calcium levels and reduces the peak of the calcium signal elicited by FMLP at concentrations ranging from 10 ° to 10 M. 

Living cells visualization of membranes, lipids, proteins, DNA, RNA, surface antigens, surface glycoconjugates membrane dynamics membrane permeability membrane potential intracellular pH cytoplasmic calcium, sodium, chloride, proton concentration redox state enzyme activities cell-cell and cell-virus interactions membrane fusion endocytosis viability, cell cycle cytotoxic activity... 

Selected entries from Methods in Enzymology [vol, page(s)] Chelation, 238, 74, 76, 297 buffers [for analysis of exocytosis, 221, 132 preparation, 219, 186 modulation of cytosolic buffering capacity with quin2, 221, 159] fluorescence assay, 240, 724-725, 740-742 fluorescence imaging, 225, 531 238, 303-304, 322-325, 334-335 free intracellular levels after bacterial invasion, 236, 482-489 free calcium in solutions for membrane fusion analysis, calculation and control, 221, 149 homeostasis mechanisms, 238, 80 hormonal elevation, 238, 79 inositol phosphate effect on release, 238, 207 determination of cytosolic levels [computer methods, 238, 73-75 with fura-2, 238, 73, 146 with indo-1, 238, 298, 316-317 with quin-2, 238, 297] hormone effects, 238, 79 ionomycin effects, 238, 79 membrane depolarization effects,... 

Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl -A (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can he blocked by botulinum toxin. Acetylcholine s action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.

The accompanying depolarization of the membrane at the synaptic ending permits a rapid inflow of calcium ions through a voltage-gated calcium channel.444 560 Within less than 0.1 ms the transient increase in intracellular [Ca2+] triggers the release of the contents of the vesicles. About four calcium ions are needed to release one clathrin-coated vesicle (Fig. 30-20A,B). The membrane fusion required for transmitter release involves cytoskeletal proteins of the synaptic endings as well as specialized proteins that are present in the membranes of the synaptic vesicles (Table 30-6). In fact, every step in the cycle depends upon specialized proteins.387... 

The depolarization that accompanies the action potential induces an increase in membrane permeability to calcium ions. A large inward electrochemical gradient exists for calcium and it moves into the terminal. The calcium that enters the terminal activates enzymes that cause the attachment of some of the vesicles to releasing sites on the terminal membrane, membrane fusion, and the release of the vesicular contents into the synaptic cleft. Transmitter release is terminated by the removal of calcium from the terminal cytoplasm, either via a calcium pump, which pumps it out of the cell, or by uptake into the endoplasmic reticulum or into mitochondria. 

Influx of calcium causes fusion of vesicle with cell membrane... 

Brunger AT. Structural insights into the molecular mechanism of calcium-dependent vesicle-membrane fusion. Curr. Opin. Struct. Biol. 2001 11 163-173. 

Melia TJ Jr. Putting the clamps on membrane fusion how com-plexin sets the stage for calcium-mediated exocytosis. FEES Lett. 2007 581 2131-2139. 

This membrane fusion process (outside the brain) is known to involve thousands of single membrane units, previously thought of as vesicles, assembled into units that have been termed "boutons". We have examined the EM texture of the boutons and found that they are in fact a cubic phase. The synaptic signal transmission can take place as frequently as hundreds of times per second. A fusion process involving a hyperbolic membrane can be well controlled, and the calcium ion influx - which induces fusion - is expected to change the conformation of the cubosome surface membrane from its planar bilayer conformation to the fusogenic saddle-saddle conformation. (It is known phase transitions of membrane lipids can occur when exposed to calcium, e.g. [40]). 

SNAP-25 and syntaxin form a stoichiometric complex. This complex can bind one molecule of VAMP with high affinity. This trimeric SNARE complex is stable in sodium dodecylsulfate (Chapman et al., 1994 Hayashi etal., 1994, 1995). In the process of neuroexocytosis, SNARE complex formation precedes the recruitment of cytosolic and membrane protein components required for fusion of the lipid bilayers (Rothman, 1994 Sollner, 1995). It is likely that NSF-mediated hydrolysis of ATP provides energy for priming the neuroexocytotic apparatus. The primed system is now ready to trigger exocytosis upon calcium influx into the synapse. It is not yet established if the last step of neurotransmitter release takes place via a fusion pore or through a complete membrane fusion with lipid intermixing (Monk and Fernandez, 1994 Bruns and John, 1996). 

Many studies show that divalent cations promote membrane fusion (27, 28, 29) and thereby may initiate attachment of granules to the insides of plasma membranes during secretion. Actually these ideas (i.e. enzyme activation and fusion of lipoid membranes by calcium), are not mutually exclusive since it is possible that calcium initiates more than one intracellular change to trigger the secretory process. 

J. Wilshut, N. Duzgunes, R. Fraley, D. Papahadjopoulos, Studies on the mechanism of membrane fusion kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents. Biochemistry, 1980, 19, 6011-6021. 

Yoshihara, E. Nakae, T. Quantitative measurement of membrane fusions induced by calcium and polyethylene glycol using the porin function. FEBS Lett. 1984, 166, 49-52. 

Fig. 6.4 Model for protein-mediated membrane fusion and exocytosis. a The release of acetylcholine from the vesicles is mediated by a series of proteins collectively called SNARE proteins. Synaptotagmin is the neuronal Ca " receptor detecting C entry. Synaptobrevin (i.e. vesicle-associated membrane protein, VAMP) is a filament-like protein on the vesicle, b During depolarisation and calcium entry, synaptobrevin on the vesicle unfolds and forms a ternary complex with syntaxin/SNAP-25. This process is facilitated by phosphorylation of synapsin, also present on the vesicle membrane, c Assembly of the ternary complex forces the vesicle in close apposition to the nerve membrane at the active zone with release of its contents, acetylcholine. The fusion is disassembled, and the vesicle is recycled. (From Martyn 2005, p 864 copyright Elsevier)...

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