The presynaptic membrane has specific sites called active zones where small vesicles containing nonpeptide neurotransmitters can fuse. To fuse with the presynaptic membrane at an active zone, a small vesicle must dock and undergo a priming process. After priming, the vesicle can release its transmitter into the synaptic cleft when there is an increase in local cytoplasmic calcium concentration. Approximately 25 proteins are involved in docking, priming, and fusion. These proteins can be cytosolic, associated with the vesicle membrane, or the presynaptic plasma membrane. The functions of most of these proteins are not fully understood, but there has been significant progress in understanding the molecular details of transmitter release.
Neurotransmitter release, like other exocytotic processes, involves SM (sec1/Munc18-like) and SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins. Vesicle-associated SNARE proteins (v-SNARES) in the vesicle membrane and target membrane-associated SNARE proteins (t-SNARES) in the presynaptic plasma membrane interact in a zipper-like manner with the help of SM proteins. This interaction brings the vesicle membrane and the presynaptic plasma membrane close together before fusion. The SNARE proteins are targeted by various botulinum toxins, which disrupt synaptic transmission, demonstrating their crucial role in this process. However, they do not bind calcium, so another protein must serve as the calcium sensor that triggers the fusion event. Evidence suggests that synaptotagmin, specifically its second cytoplasmic domain, is the calcium sensor. Synaptotagmins exhibit variations in kinetics, and different synaptotagmin family members act as calcium sensors for vesicular fusion in different brain regions. Therefore, the expression of synaptotagmin genes may contribute to adapting the kinetics of vesicle release and tailoring the specific characteristics of synaptic transmission in various regions of the central nervous system.
Calcium channels are present in the active zone membrane near the docked vesicles. Upon opening, these channels create a microdomain of high calcium concentration at the active zone. This brief local increase in calcium concentration allows synaptotagmin to bind calcium rapidly, triggering the fusion of the docked vesicle and subsequent release of its neurotransmitter. Despite involving multiple steps, vesicular release at synapses is exceptionally fast due to the close proximity of the molecular components involved. The time from calcium influx to vesicle fusion is approximately 0.2 msec.
3.6 mV 47 msec A 1 mV 20 msec B •Fig. 6.5 A, SpontaneousmEPPsrecordedataneuromuscularjunctioninafiberoffrogextensordigitorumlongus.B, EPPsevokedbynervestimulationunderlow-[Ca++]conditions,whichreducetheprobabilityoftransmitterrelease.Thesmall-amplitudeEPPsevokedundertheseconditionsvaryinamplitudeinastep-likemanner,wherethesizeofthestepisequaltothesmallestEPP,whichinturnequalsthesizeofthemEPPs.(Notethatintheseconditionsthestimulusoftenfailstoevokeanyresponse,asindicatedbyaflatresponse.)(A, DatafromFattP,KatzB.Nature 1950;166:597;B, datafromFattP,KatzB.J Physiol 1952;117:109.)
The small vesicles that contain nonpeptide neurotransmitters can fuse with the presynaptic membrane only at specific sites called active zones. To become competent to fuse with the presynaptic membrane at an active zone, a small vesicle must first dock at the active zone and then undergo a priming process. Once primed the vesicle can fuse and release its transmitter into the synaptic cleft in response to an increase in local cytoplasmic [Ca++]. On the order of 25 proteins may play roles in docking, priming, and fusion. Some of these proteins are cytosolic, whereas others are proteins associated with the vesicle membrane or the presynaptic plasma membrane. The functions of most of these proteins are incompletely understood; however, knowledge of the molecular details of transmitter release has increased dramatically in recent years.
As with other exocytotic processes, neurotransmitter release involves SM (sec1/Munc18-like) and SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins: v-SNARES in the vesicle membrane and t-SNARES in the (target) presynaptic plasma membrane. Zipper-like interactions between synaptobrevin (a v-SNARE) and syntaxin and SNAP-25 (both are t-SNARES) with the assistance of SM proteins bring the vesicle membrane and the presynaptic plasma membrane close together before fusion. The SNARE proteins are targets for various botulinum toxins, which disrupt synaptic transmission, thus demonstrating their critical role in this process. Nevertheless, they do not bind Ca++ , so another protein must be the Ca++ sensor that triggers the actual fusion event. Evidence indicates that a synaptotagmin protein is almost certainly the Ca++ sensor and, even more specifically, that the second of its two cytoplasmic domains contains the Ca++ binding site. Interestingly, synaptotagmins differ in their kinetics, and brain regions vary as to which synaptotagmin family member acts as the Ca++ sensor for vesicular fusion. Thus, differential expression of synaptotagmin genes in neurons may be a mechanism to adapt the kinetics of vesicle release and thereby tailor the specific characteristics of synaptic transmission to the functional needs of each CNS region.
Calcium channels are located in the active zone membrane at sites adjacent to the docked vesicles. When they open, a small area of high [Ca++], a microdomain is created at the active zone. This local high concentration (which lasts for less than a millisecond), allows the rapid binding of Ca++ to synaptotagmin, triggering the fusion of a docked vesicle and allowing release of its neurotransmitter. Despite the multiple steps involved, the process of vesicular release at a synapse is extremely rapid because of the close proximity of the molecular apparatuses involved to each other. Indeed, the time from Ca++ influx to vesicle fusion is about 0.2 msec.