Ukr.Biochem.J. 2011; Volume 83, Issue 2, Mar-Apr, pp. 53-64

Exocytotic steps in cell-free system after cholesterol deprivation in synaptosomal plasma membranes and synaptic vesicles

V. P. Gumenyuk, I. O. Trikash

Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;

Using a cell-free system we investigated a specific role of cholesterol in exocytotic processes. To modulate the cholesterol content in membrane methyl-β-cyclodextrin was used as a cholesterol binding agent. The experimental conditions for cholesterol depletion from synaptosomal membrane structures were determined and depended on methyl-β-cyclodextrin concentration, time and mediums temperature. The role of cholesterol was studied on the stages of synaptic vesicles docking and Ca2+-stimulated fusion which are the components of multivesicular compound exocytosis. Using­ dynamic light scattering technique we have found that after cholesterol depletion from synaptic vesicles the process of their aggregation (docking) remains unchanged.
It was found that the rate of calcium-triggered fusion of synaptic vesicles depends on the membrane level of cholesterol. The decreasing level of synaptosomal plasma membrane cholesterol by 8% leads to suppression of the Ca2+-dependent membrane fusion with synaptic vesicles. But, under 25% reduction of plasma membrane cholesterol the level­ of membrane merging with synaptic vesicles did not differ from control; probably this is due to changes in physical properties of lipid bilayer and/or disturbances in function of membrane proteins driving this process.
In cholesterol depleted synaptosomes the exocytotic release of glutamate stimulated by calcium was decreased by 32%. Obtained data suggest that the cholesterol concenration in synaptosomal plasma membranes or synaptic vesicles is the crucial determinant for synaptic transmission efficiency in nerve terminals.

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