Lecture 3: Neurotransmitter Release Machinery Flashcards
presynaptic neuron role
providing synaptic vesicles
- any given AP causes a quantal number of vesicles to fuse; train of APs causes more to fuse, but any one given AP does not cause a lot of NT release
postsynaptic neuron role
responds to NT message
- neurons can be both pre- and post-synaptic, just not at the same synapse
synaptic cleft role
NT diffuses from pre to post due to simple concentration gradient
- protein dense zone that helps organize and link the synapse, provides scaffolding
what is the tripartite synapse composed of
- presynaptic neuron
- postsynaptic neuron
- astrocyte
astrocyte role in synapse
- end feed surround synapse to insulate
- participate in NT and ion recycling (ex: get glutamate out of the synaptic terminal and turn back to glutamine)
- lots of Ca2+ signaling
proteins involved in NT loading
- transmitter transporters
- proton pump
proteins involved in mobilization
synapsins
proteins involved in docking
RIM coordinating complex (RIM, Rab 3 / Rab 27, RIM-BP, Munc13)
proteins involved in priming
- SNARE complex (synaptobrevin, syntaxin, SNAP-25, Munc18)
- Munc13
proteins involved in fusion
- synaptotagmin
- complexin
proteins involved in coating
- Clathrin
- AP-2
- Stonin
- AP180
- NSF
- SNAP
proteins involved in budding
- dynamin
- amphiphysin
proteins involved in uncoating
- clathrin
- auxilin
- Hsc-70
- endophilin
- synaptojanin
what is the regulated secretory pathway
a process of exocytosis in which soluble proteins and other substances are initially stored in secretory vesicles for later release in response to a signal
what is the constitutive secretory pathway
after vesicle leaves the Golgi apparatus and ER, there are things that can help convert precursors into the new signal inside the vesicle
- no need for a signal
main differences between neurotransmission and the typical regulated secretory pathway
- product that goes into these regulated secretory pathways is already in its complete form
- vesicle at membrane will not fuse without a signal
advantage of chemical synaptic transmission as an adaptation of the regulated secretory pathway
allows it to be very fast, and very reliable
2 essential adaptations in regulated secretory process that support the speed of synaptic transmission
- small, clear core vesicles
- local vesicle recycling in the endosome – avoids having to transport filled vesicles from Golgi
characteristics of small, clear core vesicles
- greatly simplified vesicle contents
- packaged up w/ small molecule NTs
- less proteins, so NTs must be in the vesicle in their ready forms
first step of chemical synaptic transmission as an adaptation of the regulated secretory pathway
delivery of synaptic vesicle membrane contents to the presynaptic plasma membrane
second step of chemical synaptic transmission as an adaptation of the regulated secretory pathway
endocytosis of synaptic vesicle membrane components to form new synaptic vesicles directly
third step of chemical synaptic transmission as an adaptation of the regulated secretory pathway
endocytosis of synaptic vesicle membrane components & delivery to endosome
fourth step of chemical synaptic transmission as an adaptation of the regulated secretory pathway
budding of synaptic vesicle from endosome
fifth step of chemical synaptic transmission as an adaptation of the regulated secretory pathway
loading of NT into synaptic vesicle
sixth step of chemical synaptic transmission as an adaptation of the regulated secretory pathway
secretion of NT by exocytosis in response to an AP
perisynaptic zone
important in vesicle signaling
- endocannabinoids have most receptors in this zone (weird)
three phases of NT release
- docking
- priming
- fusion
what forms the SNARE complex
V SNAREs and T SNAREs form a long alpha helical domain; when the two SNAREs come together, they zipper
- going from high energy –> low energy (lying flat) zipper states requires Ca2+
what are V SNAREs
vesicular SNAREs (synaptobrevin)
what are T SNAREs
target snares (SNAP-25)
active zone protein in SNARE complex
Munc18
exocytosis gatekeepers
synaptotagmin-1 and complexin
synaptotagmin-1 function
Ca2+ sensor
complexin basic function in priming
responsible for signal-dependent release aspect of this process
RIM coordinating complex function
coordinates shape of the vesicle, makes sure that all the Ca2+ and vesicles are at the active zone together
Calyx of Held significance
- found in mammalian CNS auditory pathway
- massive presynaptic terminal
- can voltage or current clamp both pre- and post-synaptic neurons
docking
getting the vesicle to the active zone and holding it there
how to measure docking
manually count docked vesicles using microscopy
what is RIM bound to
plasma membrane
what are Rab 3 and Rab 27 bound to
vesicle membrane
interaction between RIM and Rab 3 / Rab 27
holds the synaptic vesicle close to the plasma membrane to allow for priming to occur
Han et al (2011) experiment contribution to RIM knowledge
used microscopy to measure quantitative number of docked vesicles upon RIM KO, and found that the KO had fewer number of docked vesicles
how does Munc13 function in docking
after RIM and Rab bind each other and hook the vesicle up to the Ca2+ channel, Munc13 comes in and binds to the TIM coordinating structure right after the complex forms
- doesn’t participate in docking
how does RIM activate Munc13
breaks up the Munc13 dimers, allows single Munc13 to bind to RIM-coordinating complex
significance of Kaeser et al (2011) experiment to docking
determined that RIM KO on hippocampus neurons in a dish severely diminishes synaptic transmission
priming
prepares the vesicle for triggering of Ca2+ dependent fusion (and NT release)
what is the SNARE complex formed by
vesicular SNARE (synaptobrevin) coming together with plasma-membrane target SNAREs (SNAP-25 and syntaxin)
how is priming RIM dependent
RIM activates Munc13 by interfering with Munc13 homodimerization
Munc13 role in priming
sets syntaxin to its open conformation
Munc18 role in priming
binds to syntaxin, bringing the SNARE complex together
does Munc13 affect docking?
no, but it does affect neurotransmission
what did Varoqueaux et al (2002) experiment contribute to Munc13 research
found that Munc13 KO severely impaired excitatory, inhibitory, and spontaneous (with alpha-lacrotoxin) neurotransmission
alpha-lacrotoxin role in Munc13 experimentation
it normally causes spontaneous synaptic release, so it was a tool to cause the fusion of vesicles even without Ca2+
does Munc13 affect fusion?
no
Augustin et al (1999) experiment significance to Munc13 research
determined that Munc13 does not affect fusion
- injected alpha lacrotoxin (spontaneously causes fusion to occur), and found that fusion still happens with Munc13 KO
how did they reason that Munc13 is important for priming?
they ruled out that it’s not important for docking or fusion, so therefore it must be important for priming
difference between tetanus & botulinium effects
tetanus = rigidity, botulinium = paralysis
- each toxin cleaves SNARE, but effect depends on whether it is cleaved in an excitatory (botulinium) or inhibitory (tetanus) neurons
where is botulinium taken up
motor neurons
where is tetanus taken up
interneurons in the spinal cord
when do docking and priming occur
prior to AP
Munc18 and Munc13 similarities
both cause the SNARE complex to come together
complexin function in invertebrates
- inhibits tonic release (allows for Ca2+ dependent release)
- makes stimulus evoked release even stronger
Trimbuch & Rosenmund (2016) experiment significance to complexin
found that complexin absence causes more spontaneous release, and less synchronous release
Martin et al (2011) experiment significance to complexin
found that different regions of complexin show different roles: getting rid of certain parts gets rid of inhibition, and another part gets rid of all effects
complexin function in mammals
enhances fusogenicity of vesicles for spontaneous & evoked release
- more spontaneous activity for both excitatory & inhibitory synapses when complexin is present
Trimbuch & Rosenmund (2016) experiment significance to complexin in mammals
in mammals, complexin facilitates fusion by lowering the energy barrier needed to go from a primed to fused state
Trimbuch & Rosenmund (2016) experiment significance to complexin in invertebrates
in invertebrates, complexin facilitates evoked release by lowering the energy barrier needed to go from a primed to fused state, but inhibits spontaneous release
first step of fusion
free SNAREs on vesicle and plasma membrane
second step of fusion
SNARE complexes form as vesicle docks
third step of fusion
synaptotagmin binds to SNARE complex
fourth step of fusion
entering Ca2+ binds to synaptotagmin, leading to curvature of plasma membrane, which brings membranes together
fifth step of fusion
fusion of membranes leads to exocytotic release of NT
requirements for synaptic vesicle fusion w/ the plasma membrane & resulting exocytosis of NT
- elevation of Ca2+ at the vesicle (results from extracellular Ca2+ entry during depolarization)
- activation of synaptotagmin (the Ca2+ sensor)
- completion of the SNARE complex
synapsin
one of the presynaptic proteins responsible for moving vesicles from the pool to where they need to be docking
Kaeser et al (2011) experiment significance
almost all vesicles are near voltage-gated Ca2+ channels
- synapsin KO causes Ca2+ channels to no longer be found in the active zone
- adding back RIM 1 alpha causes Ca2+ channels to stop popping back up
what happened after adding RIM RNA that is deficient in PDZ binding domain to a knockout condition?
prevents the rescue we saw from adding back RIM 1 alpha
RIM PDZ binding domain
where Ca2+ binds to RIM
what is RIM crucial for
co-localization of Ca2+ to active zone, and to the particular binding domain
how did Kaeser et al determine that RIM KO did not impact cell functioning?
RIM KO did not affect bassoon function
bassoon function
moving vesicles from reserve pool to docking
what does Ca2+ binding to C2A and C2B regions of synaptotagmin trigger
fusion
what the activation of synaptotagmin triggering fusion result in
removal of complexin from SNARE complex
what is the main thing that causes the delay for chemical transmission
fusion
what is Munc18 essential for
successful synaptic vesicle fusion
significance of Verhage et al (2000) experiment
showed the Munc18 KO in neocortex & NMJ causes loss of spontaneous and synchronous activity
- loses the ability for alpha lacrotoxin to fuse
first hypothesis for Munc18 function
Munc18 mediates lipid mixing during vesicle membrane - plasma membrane
second hypothesis for Munc18 function
Munc18 catalyzes or nucleates SNARE complex formation in the active zone
third hypothesis for Munc18 function
Munc18 organizes the SNARE complex around the fusion site
