The role of protein phosphorylation in the switching between modes of synaptic vesicle release

Abstract

Among secretory systems, the nerve terminal is unique in its capability for sustained release of vesicular contents in response to high rate stimulation and attention was focused on the presynaptic nerve terminal - by employing synaptosomes - for understanding the mechanisms occurring in synaptic vesicle exocytosis. Synaptic vesicles have rapid recycling capabilities and this in part may be due to a kiss-and-run mode of neurotransmitter release. To investigate the mode of exocytosis synaptosomes were treated with various drugs and both glutamate and FM2-10 dye release were measured. At the same time, the effect that such drug treatments may have on evoked changes in the intracellular calcium was determined using Fura 2 measurements, Acute bafilomycin Al treatment of synaptosomal preparation indicated that the stimuli employed
induced only one round of vesicle exocytosis. This meant that glutamate and FM2-10 dye release could be directly compared. Previous results had suggested that changes in the intracellular calcium may be involved in causing a switch
between a full fusion mode and a kiss-and-run mode of exocytosis. To investigate whether L-type calcium channels could regulate the fusion mode, these channels were inhibited by nifedipine and it was discovered that this had no effect on glutamate release but caused a large increase in FM2-10 dye release.
This indicated that blockade of these channels led to a switch from kiss-and-mn to full fusion of a population of synaptic vesicles. Intriguingly, specific activation by phorbol ester of calcium-dependent protein kinase C also led to switch from a kiss-and-run mode to a full fusion mode of exocytosis. This and earlier data indicated that protein phosphorylation and calcium levels play a role in discerning the mode of synaptic vesicle exocytosis, but did not reveal which proteins might be involved in directly modulating the properties of the fusion pore involved in exocytosis. Investigations were carried out to see if dynamins or myosin 2 could regulate the fusion pores of synaptic vesicles since these proteins have been hypothesised to play such a role in non-neuronal exocytosis.
Inhibition of dynamin 1 and 2 by dynasore revealed that for some stimuli, such proteins may play a role in the quick closure of the fusion pore. Likewise, inhibition of myosin 2 by blebbistatin also revealed that with some stimuli this protein might also regulate the kinetics of the fusion pore opening. Intriguingly, there were some differences between these two inhibitors and the effects that they had on specific stimuli. Thus, it would appear that, under different conditions, dynamin 1 or 2 or myosin 2 may be important in the closure of the synaptic vesicle fusion pore during exocytosis. Several neuronal defects are
known to occur on account of faulty proteins. Hence, detailed future studies on protein-protein interactions between various phosphorylated proteins that contribute to the opening and closing of the fusion pores in synaptic vesicle exocytosis could lead to important findings that might aid in the understanding of certain neuronal diseases.