7.2 Triggering systems
In neurons, the stimulus for vesicle release is usually a depolarisation (action potential) that causes calcium to enter the nerve cell through voltage-gated calcium channels. The rise in intracellular Ca2+ concentration,[Ca+], causes the vesicle to fuse with the plasma membrane and a large amount of neurotransmitter is then released. Although the SNARE complex constitutes the essential fusion machinery of the synaptic vesicles, it is unclear exactly how fusion is triggered by calcium ions. Two elements appear to be important, synaptotagmin and CaM kinases, which are a family of kinases whose activity is critically dependent on the local [Ca2+ ](see Box 5).
Where is synaptotagmin located?
It is present on the membrane of the synaptic vesicle and briefly on the plasma membrane, after the vesicle has fused to release its cargo (see Figure 26).
Synaptotagmin is most probably the major calcium sensor that mediates membrane fusion at the synapse. It has a large cytoplasmic calcium-binding domain, and it has been suggested that calcium binding to synaptotagmin induces conformational changes that cause oligomerisation (i.e. aggregation of a few molecules) leading it to interact with components of the plasma membrane and thence to the assembly and clustering of the SNARE complex.
Box 5 CaM kinases (Ca2+–calmodulin-dependent kinases)
The CaM kinases are a group of kinases that are highly sensitive to changes in intracellular [Ca2+]. Moreover they can display a ‘molecular memory’. CaM kinases are normally inactive, but are activated by a rise in [Ca2+]. At this point they autophosphorylate themselves, which locks them into an active conformation. Consequently they remain active for a while, even after [Ca2+] falls.
The kinases also act on other proteins – different CaM kinases have different substrate specificities. Eventually phosphatases remove the phosphate groups on the CaM kinase and the enzyme becomes inactive again. This means, however, that the effect of a Ca2+ signal persists longer than the signal itself.
In nervous tissue, a member of the CaM kinase family known as CaM kinase II is present at very high concentration. An important property of CaM kinases is their ability to integrate and decode Ca2+ pulses. As each action potential reaches the nerve terminal it opens the voltage-gated calcium channels, thus producing a pulse of intracellular Ca2+. An increase in the rate of action potentials produces a corresponding increase in the rate of Ca2+ pulses. Because of their molecular memory, the CaM kinases can integrate this signal over time. Moreover, because of the positive feedback in their action the kinase activity of CaM kinases is highly dependent on [Ca+ ]– small changes in [Ca2+ ] produce big changes in kinase activity. One function of CaM kinase II is to phosphorylate synapsin, a protein that controls the interaction of synaptic vesicles with the cytoskeleton and hence whether they are free to bind to the plasma membrane (Figure 42). A variety of methods using fluorescent tracers has been used to elucidate the mechanisms of vesicle fusion (Box 6).
Calcium-dependent signalling controls secretion in many other cell types. Can you recall another example?
The rate of release of insulin by b cells in the pancreas is regulated by intracellular [Ca2+] – see Section 2.4.
Box 6 Investigating exocytosis and endocytosis
Release of neurotransmitters from small synaptic vesicles can be visualised with a fluorescent dye called FM2–10. This dye is not fluorescent in solution but it becomes so after binding to cellular membranes. Thus, when a synaptic terminal is exposed to FM2–10, the external membrane becomes fluorescent. If the neuron is stimulated at this time, the membrane of small synaptic vesicles fuses with the plasma membrane where they encounter the FM2–10. When this vesicular membrane is recycled by endocytosis the synaptic vesicles within the nerve terminal become fluorescent. If the externally added FM2–10 is removed from the medium, the plasma membrane will lose its fluorescence but the internal synaptic vesicles will remain fluorescent. Subsequent stimulation of the nerve terminal thus allows measurement of the rate of fusion of synaptic vesicles at the synaptic junction, as shown in Figure 43.
Most studies of neurotransmitter release use electrophysiological measurements of the postsynaptic response to presynaptic events. However, it is also possible to follow presynaptic activity directly by measuring changes in membrane capacitance. When a synaptic vesicle or secretory granule from a non-neuronal cell fuses with the plasma membrane during exocytosis, the surface area of the secreting cell increases. Conversely, the surface area decreases when membrane is retrieved by endocytosis. These changes in the cell surface can be followed by measuring the electrical capacitance of the cell membrane (Figure 44).