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Section I: Cellular and Molecular Neurobiology 10. Transport and the Molecular Mechanism of Secretion Part 4 of 5 Jack C. Waymire, Ph.D.

Secretory Mechanism

As shown in Figure 10.7, the first event that must occur (with the exception of neuropeptide neurotransmitters) is the filling of vesicles with neurotransmitter through specific neurotransmitter uptake (NT Uptake). This uptake will be covered in subsequent chapters that discuss each of the specific neurotransmitters.

The vesicles remain in reserve until needed for secretion. When needed for secretion, a translocation occurs, which is also referred to as mobilization. The vesicles move to a region of plasma membrane called the active zone. The active zone is the release site and is characterized by the appearance of dense material adjacent to the plasma membrane. The influx of Ca²⁺ is believed to increase translocation by increasing the Ca²⁺ dependent phosphorylation of a vesicle binding protein termed synapsin. The theory is that Ca²⁺ dependent phosphorylation of synapsin frees the vesicles from binding to actin microfilaments. The vesicles then bind to the active zone of the plasma membrane, where they are in position to undergo release of their neurotransmitter.

The association of the vesicle with the plasma membrane is termed docking and serves to prime the vesicle for secretion. The docking is believed to occur through the binding of proteins in the vesicle membrane to proteins in the plasma membrane. Several of these proteins have been discovered because they are targets for clostridia bacterial toxins that block synaptic transanimation. Several of these toxins and the proteins they detect are shown in Table I. The toxins are so toxic that a single molecule can poison a whole nerve terminal. One of the synaptic vesicle proteins is VAMP, and two of the synaptic plasmal membrane proteins are syntaxin and SNAP-25.

A third plasma membrane protein, n-sec-1, is important because its loose association with the plasma membrane prevents the binding of the neurotransmitter vesicle proteins until n-sec-1 is displaced (the mechanism of n-sec-1 displacement is currently not understood). This and subsequent steps in the secretory process are shown in Figure 10.8. The vesicle and plasma membrane proteins are hypothesized to complex with one another upon the displacement of n-sec-1 to form a "trimeric complex" (SNAP-25, syntaxin and VAMP). This three-member complex has been isolated, intact, from the nerve endings of animals. This association of the proteins initiates fusion. Vesicles at this stage are primed for release.

The final stage of release, also shown in Figure 10.8, is the fission of the membrane at the point of contact between the vesicle and the plasma membrane. Exocytosis of neurotransmitter into the synaptic cleft occurs when this fission takes place. This step is Ca²⁺ stimulated, but the mechanism of the Ca²⁺ trigger is unknown. One hypothesis is that a vesicle protein called synaptotagmin binds Ca²⁺ to initiate fission. Support for synaptotagmin, as the Ca²⁺ sensor, is that it possesses two binding sites for Ca²⁺. Additional evidence comes from studies of mice in which synaptotagmin has been knocked out. In these mice fast Ca²⁺-triggered synaptic vesicle exocytosis is severely limited. Many aspects of the fusion-fission mechanism remain to be understood, including: what causes the dissociation of n-sec-1 from the complex, how Ca²⁺ functions in the release process and how all the proteins that are involved in release become reassociated with the proper membrane following release as the vesicle membrane is recycled.