Neurotransmitters, neuropeptides, and neurotransmission Flashcards
Spell out four main differences between classical and peptide transmitters.
Synthesis:
Classical t. = in cytoplasm
Peptide t. = translation of mRNA + Golgi apparatus process
Storage:
Classical t. = synaptic vesicles
Peptide t. = secretory vesicles (dense core)
Release site:
Classical t. = active zone
Peptide t. = unspecified
Types of neurotransmission mediated:
Classical t. = fast and slow
Peptide t. = slow
By what channel type is mediated fast neurotransmission? Slow?
Fast: ligand-gated ion channels
Slow: g-prot linked receptors
List 9 classical neurotransmitters.
ACh
Glu
GABA
Glycine
Dopamine
Norepinephrine
epinephrine
Serotonin
Histamine
Name the anatomical differences between slow and fast transmitters.
Modulatory (slow) neurotransmission is mainly originating from the midbrain and project widely through the cortex.
What is Dale’s hypothesis? Is it always true?
Neurons use only 1 type of neurotransmitter. Many exceptions, e.g. GABA and glycine can be released from the same synaptic vesicle
List three characteristics of ATP in regard to neurotransmission.
Slow and fast ligand-gated channels
Can be transported into vesicles
Most likely released from secretory granules
What does a neuron always need to release transmitters from a synaptic vesicle?
A vesicular transporter to concentrate the transmitter into a vesicle
What are the two main types of neurotransmitter transporters?
Plasma membrane transporter
Vesicular transporter
Name the 4 types of vesicular transporters
Vesicular Monoamine Transporter (VMAT1 and VMAT2)
Vesicular Acetylcholine Transporter (VACHT)
Vesicular GABA and Glycine Transporter (VGAT)
Vesicular Glutamate Transporter (VGLUT)
True or false: 3 families or transporters were co-opted independently for synaptic vesicles.
False, three independent evolutionary events. VGAT and VGLUT are present in Cnidaria; VMAT/VACHT was coopted later. VATP is actually the oldest; predates synaptic vesicles
What is the formula of synaptic strength and what are its variables?
M = NPQ
M: synaptic strength
N: # of release sites
P: probability of release
Q: change in postsynaptic membrane potential due to one single synapse
How is a change in synaptic strength usually probed?
One often looks at miniature excitatory post synaptic currents (minis) that are observed in the absence of an action potential and are due to the spontaneous release of one synaptic vesicle
How is P or N affecting postsynaptic minis? Q?
P or N: increase in freq.
Q: increase in amplitude of mini
Why do transporters require energy?
Because they perform work. Energy can be ATP or trading ions along their electroconcentration gradients
What can explain that transporter overexpression leads to increase in Q?
Vesicles leak (leaky bathtub model)
How can a change in Q (e.g., levels of Vglut) affect mini freq.?
Same number of synaptic vesicles will bind, but some of them could be empty, leading to a lower freq. postsynaptic recording of minis
True of false: loss of transporters leads to changes in synaptic vesicle release.
False
How can co-expression of transmitters be implemented (3)?
Storage of two transmitters in same vesicle
Two separate populations of synaptic vesicles in same release site
Separate release sites with distinct populations of synaptic vesicles
Explain the synergy between VACHT and VGLUT3.
Positively charged Ach builds up in the vesicle ->harder to pump in H+ (ATP) ->less ACh can be exchanged for H+ (VACHT)
Pumping in negatively charged Glu (VGLUT3) -> vesicle less positively charged ->ATP pump works better -> VACHT works better
True or false: the Vesicular ATPase cares about both the charge and pH of the vesicle
True.
What is the most common occurence of transporter synergy?
VGLUT and Zn: role of Zn in facilitation Vglut transport would be to dissipate the charge difference
Can dopamine be converted to norepinephrine?
True.
Why is removal of neurotransmitters a critical step in neurotransmission?
Fast: allows for high freq. signaling
Slow: prevents undesired lasting modulatory effects
Why is glutamate taken up by glial cells instead of neurons themselves?
Faster for astrocytes to reuptake.
How is the synthesis of neuropeptides and classical transmitters differentiating?
Neuropeptides: made from protein precursors encoded by genes
Classical transmitters: synthesized by enzyme in the cytosol
In what vesicle type are neuropeptides stored?
Dense core.
Why do neuropeptide precursors have cleavage sites?
To release active peptide.
What is the signal sequence on neuropeptide precursors necessary for?
Entry to ER.
Which statement is false?
1. Most neurons that secrete neuropeptides also have classical transmitter.
2. Can be multiple neuropeptides in same neuron.
Both true.
Why are neuropeptides made as precursors?
Small peptides cannot be inserted into the ER co-translationally
Where does cleavage of neuropeptides occur?
Trans-Golgi network (TGN)
What is the secretory pathway of neuropeptides?
Ribosomes + rough ER co-translation: translocation, signal seq. cleavage, folding -> Golgi: glycosylation -> Trans-Golgi network (TGN): endoproteolytic cleavage, sorting into secretory vesicles
True or false: transmembrane proteins are sorted in dense core vesicles, like neuropeptides.
True, BUT storage would occur at two distinct time points.
Furin and Prohormone convertases (PCs) cleave the peptides from their precursos. Where are they respectively recruited?
Furin cleaves in the TGN; PCs cleave in immature secretory granules.
Where does cleavage of the neuropeptide from its precursor (endoproteolytic cleavage) occur?
TGN
How can levels of neuropeptides be regulated?
Expression and release.
Why cant neuropeptides be released at high freq. like classical transmitters?
Bc. packaging at the TGN through sorting does not allow for efficient formation of dense-cored vesicles at synapses
Time for new DCV to arrive at synapse (hours)
Under which excitation conditions are DCVs released?
At high freq.
What is the difference between G protein-linked receptors, seven transmembrane receptors, and metabotropic receptors
no diff., all the same
True or false: Ga is the G protein.
True.
Specify what the three major types of G prot. are and do (3).
Gs : increase adenylate cyclase; make cAMP
Gi : decrease adenylate cyclase, act directly on ion channels
Gq : increase phospholipase C
Name 2 G protein effectors and list their effects
Adenylate cyclase makes cAMP:
-reg. of PKA
-direct reg. of ion channels
Phospholipase C makes IP3 and DAG:
-IP3 releases calcium from internal stores (ER)
-DAG binds to C1 domains = GEFs activation, increase release by binding to UNC13, etc.
Ion channels can be modified 3 ways by slow neurotransmission, list them.
-Ga or Gbg binding directly to the channel
-Direct binding of second messenger to the channel (e.g. cAMP-gated channels)
-Phosphorylation of the channel by second-messenger gated kinase (e.g G protein–> phospholipase C –> Diacylglycerol –> PKC –> phosphorylation of channel.
How are slow EPSPs mediated?
Closing Resting K+ channels
OR
Opening Resting Cation channels (Na and K+)
How are slow IPSPs mediated?
Opening Resting K+ channels
What can affect the shape of the AP?
Closing Voltage Gated K channels or opening Voltage gated Ca channels
What proteins are involved in the size of PSP in fast neurotransmission?
Ligand-Gated Ion Channels; Changes in transmitter release
What mechanisms can regulate time of G protein activation?
-Desensitization of Receptor (Phosphorylation/Internalization)
-GTPases to turn off G protein
-Enzymes to Degrade Second Messengers
-Phosphatases to reverse actions of kinases
Why do release sites need to be closed to calcium channels?
Proteins mediating exocytosis of synaptic vesicle require calcium -> if entry of calcium is closer to exocytosis apparatus, process will happen faster
What analogy can help understand why fast reactions only occur with low affinity reactions?
Which is faster opening a door with a key (high affinity) or hitting a button (low affinity
What type of proteins are tethering calcium channels to synaptic vesicles?
Scaffold proteins
How is synaptic vesicle fusion different from other cellular fusion types?
calcium sensitivity and specificity for active zone
What were the four lines of neuroscience research looking for SNAREs?
Golgi-golgi fusion field
Looking for proteins on synaptic vesicles field
Toxins blocking NT release field
Yeast genetic screens for mutations blocking release field
Name two toxins that block VAMP, SNAP-25, or syntaxin.
Tetanus and botulinum
What kind of structural interation holds between the plasma membrane and the vesicle? What is the advantage?
Coiled-coil interactions, thought to provide more energy to fuse both membranes
In a single interaction between a vesicle and the plasma membrane, how many coiled coils are involved? Which SNARE involved belong to the plasma membrane? The vesicular membrane?
4 coiled coils:
3 glutamine SNAREs (Q) = on plasma membrane:
-1 from syntaxin
-2 from SNAP-25
1 arginine SNARE (R) = on vesicular membrane:
-VAMP
What tethering and templating proteins are necessary to structure the coiled-coil interaction?
Tethering by Unc-13
Templating by Unc-18
What is the function of Munc 18? Is it necessary for release?
Munc 18 forms a ‘template’ to increase correct SNARE interactions allowing for the primed state to be reached
Yes
What is unique about the template model SNARE-mediated vesicular fusion events?
Munc18 binds to syntaxin in a closed state -> preventing syntaxin to join the SNARE complex and so the complex to be primed = inhibitory effect
What is the function of Munc 13? Is it necessary for release? What is a second function of Munc13?
Munc 13 is required to disrupt Munc18 closed interactions with syntaxin and assists Munc 18 in its template function allowing for the SNARE complex to form
Yes
Tethering synaptic vesicles to plasma membrane
What is the most fundamental protein involved in bridging synaptic vesicles to calcium channels?
Munc13
Most regulatory processes recruit which protein part of the SNARE complex?
Munc13
Why could it be useful to tag Munc13 with fluorescence?
Munc 13 marks release sites
What is synaptotagmin I? What happens to release if deleted?
synaptic vesicle protein with two C2 domains (C2A and C2B) that bind Calcium in a lipid-dependent manner
no fast Calcium evoked release, but spontaneous release and asynchronous calcium evoked release remain -> but Syt I can also do spontaneous release
Asynchronous calcium dependent transmitter release requires which isoform of synaptotagmin ?
Syt VII
What calcium sensor is more important at low calcium concentration? Why?
Syt VII because it is less dependent on cooperativity to bind calcium
Which synaptotagmin isoform is more cooperative with calcium? Less?
More = Syt 1
Less = Syt 7
True or false: Syt I can do evoked and asynchronous release, and Syt 7 can only rescue asynchronous release.
True.
When does complexin bind SNAREs?
Only once when form the primed coiled coil complex
How is complexin referred to as? Why?
Fusion clamp: would MAKE the SNARE complex and release process calcium dependent, bc. evolutionarily this process is calcium independent
How would complexin prevent fusion except in presence of calcium?
Complexin would be preventing closing of the last part of coil complex (part of VAMP near the membrane) and synaptotagmin would dislocate Cpx upon binding to calcium -> finishing the complex -> vesicular fusion
How many sites for synaptotagmin are there on the SNARE complex? How many of those sites are involved with complexin? Which synaptotagmin can bind to those sites?
1 site with complexin -> on which can bind ANY synaptotagmin
1 site complexin free -> on which can bind ONLY Syt I, II and 9
Therefore, complexin might be more important for asynchronous release
Which synaptotagmins are mediating calcium evoked release (synchronous)?
Syt I, II and IX
Which domain of synaptotagmin is binding to the SNARE complex?
C2B, not the C2A
Mutation in which domain of synaptotagmin changes the affinity of release?
C2A
What happens when calcium binds to C2B?
C2B inserts into lipid bilayer (plasma membrane or synaptic vesicle), this displaces complexin and induces a conformational change to bring the coiled-coil to completion, both bringing the membranes closer and perturbing them, stimulating fusion
Is NSF necessary for fusion?
No, but it is necessary to repeat fusion bc. it unwinds the coiled coil formation of the trans-SNARE complex -> allows to vesicular and plasma membrane SNARE prot. to dissociate
Does NSFT require ATP?
Yes
What is the most important vesicle recycling (endocytosis) mechanism at synapses?
clathrin-mediated endocytosis
Match the SNARE proteins with their definition:
____ (Synaptobrevin)
-The only SNARE on the synaptic vesicle.
____
-Integral plasma membrane protein.
____
-Associated with plasma membrane through lipid attachments; not integral membrane protein (most other SNAREs are integral membrane proteins).
VAMP (Synaptobrevin)
-The only SNARE on the synaptic vesicle.
Syntaxin
-Integral plasma membrane protein.
SNAP-25
-Associated with plasma membrane through lipid attachments; not integral membrane protein (most other SNAREs are integral membrane proteins).
What is another important vesicle recycling (endocytosis) mechanism at synapses?
Bulk endosome endocytosis from which vesicles are formed intracellularly via budding, which is clathrin dependent
When are the first SV formed?
formed by endocytosis after initial insertion of proteins into plasma membrane
