Dr Anja Teschemacher Flashcards
Connexins
Gap-junction proteins 2x hemichannels (6x connexins)
Essential for depolarising cardiac muscle
ATP can pass through
Flow down concentration gradients
Forms pores between the cytosol + EC space
Enable CF epithelium to be depolarised
Pannexins
Not gap junctions
Can form pores between cytosol + EC space
ATP can pass through
Activated macrophages - recruit pannexin channels - secrete beta-interleukin
Down-regulation of EAAT1/2 channels
Connexins + pannexins
Do not show high sequence homology!!
Membrane transporters - MCT
Dependent on concentration gradient of the main substrate, or a co-substrate = drives shuffling of the molecule down its concentration gradient
Monocarboxylate transporters - lactate/pyruvate down concentration gradient
MCT1 = astrocytic
MCT2 = neuronal
Used for lactate shuffle - important in L+M
H+ = co-substrate
Membrane transporters - glutamate transporters
EAAT1/2 = astrocytic EAAT3/4 = neuronal
EAAT1/2 = down-regulated in ALS
Interleukin (P2X7-macrophages) = down-regulate EAAT1/2
Glutamate in, 3Na+ in, 1H+in, K+ out
Dependent on Na-K-ATPase
Heteroexchange
Heteroexchange - 1 transporter substrate can release another one accumulating inside the cell
DAT = Dopamine Activate Transporter
Dopamine uptake causes the release of MPP into the synaptic cleft (substrate-induced release)
Cocaine/amphetamines
Uptake through DAT
Enter synaptic vesicles via VMAT2 - collapse vesciular pH gradient, dopamine releases into the cytosol
Bind TAAR1, cause P of DAT = reversal - dopamine released into the cleft
Cross-talk between transporter regulation mechanisms
GAT1 = GABA transporter 1
Co-substrate = Na + Cl
Uses energy from the dissipation of a Na+ gradient
SCV
Model = retinal bipolar cell (goldfish)
~50nm
Mainly classical fast NTs, small amino acids, ATP
Predominantly in the CNS
Mainly act at fast ligand-gated ion channels - PSPs
Endosome derived
Electron microscopy = clear
Endocytosis = rapid local recycling
Reliant on Ca microdomains - lower affinity
4 cooperative Ca binding = very positive cooperativity
0.2ms after AP
Mainly docked at the active zone
LDCV
Model = chromaffin granule cells
~100-500nm
EM = dark + dense
Mainly outside the active zone
Catecholamines (dopamine, adrenaline, noradrenaline), amines, peptides, ATP
Golgi-derived
Predominantly in neuroendocrine cells + sympathetic terminals
2 Ca binding steps
High affinity - reliant on radial gradients
Slow endocytosis
~0.5ms after strong stimulation
Lipid bilayer fusion
Overcome 2 strong energy barriers
- Formation of a hemifusion state = outer layer vesicle forms a continuum with the plasma membrane
- Form a continuous layer with IC vesicle + EC cell - a continuous body of water
Involves energetically unfavourable steps
SNARE proteins
Synaptobrevin (vSNARE)
tSNARE: SNAP-25, syntaxin
Interact via short TMD linkers - form coiled-coil motifs
Coiled-coil formation brings the two membranes into close proximity allowing fusion to occur
SNARE motifs interact + twist together
Dock to the membrane - release energy (highly exergonic reaction); used to initiate the membrane fusion
Tetanus
Wound infection
Cleave vSNAREs of motor inhibitory interneurones
Progressive muscle spasms (due to loss of inhibition)
Botulinum toxin (Botox)
Lethal food poisoning
Cleaves SNARE complex of cholinergic neurones
mAChR = parasympatholysis (dry mouth, urinary retention etc.)
nAChR = paralysis
Can be inactivated via boiling!
Clinically - control unwanted neuromuscular disorders (nAChR) or hypersecretory disorders (mAChR)
ie. Hyperhidrosis, detrusor (bladder) hyperactivity
TIRF
Total Internal Reflection Microscopy - can be used to visualise exocytic events
Selective excitation of surface-bound fluorophores - fluorescently-tag lipid vesicles - see exocytosis of tagged vesicles from the active zone + see a subsequence transport of vesicles to RRP
Fluorescence increases closer to the footprint
Complexin
Acts as a fusion clamp + super-primes vesicles
-Binds via a central accessory helix to a groove on the SNARE complex (cannot bind to individual v/tSNAREs)
Fusion clamp = blocks the progression of SNARE zippering + fusion - releases inhibition when synaptotagmin binds Ca (C2 domain)
Super-primes SNARE complex = helps the complex to enter a highly fusiogenic state + stabilises; sensitises/prepares the complex to activation via synaptotagmin
**LINK = involved in AMPAR exocytosis during LTP (not basal trafficking)
Flash photolysis
Caged Ca - EGTA-photosensitive cage
Whole-cell patch clamp
EGTA = calcium cheater - photosensitive
UV flash = uniform [Ca] elevation, measure exocytic events
Membrane capacitance studies
Indirect measurement of vesicle exocytosis
Capacitance determines how quickly a cell’s Vm responds to a change in current
Surface area directly proportional to capacitance = increase s.a., increase capacitance
SCV ~ 2.5 fF
Voltage-Clamp - can see capacitative transients = exocytic events
Remember to use flash photolysis —cannot be measured if there is changes of conductances occurring in the membrane (ie. activation of VGCCs to stimulate exocytic events)
Problems with whole-cell patch clamp diluting IC contents
Net capacitance is a sum of exo and endo events BUT endo slower time course!
Net capacitance sum of ALL fusion events - even empire vesicles!
Calcium requirements of SCV
Retinal bipolar goldfish cell
Calcium flash photolysis + capacitance = index of exocytosis
Plotted exponential rate constant against exponential [Ca] = very steep - 4 Ca binding cooperative steps!
Domains - SCV + LDCV
SCV = Nanodomain
-Vesicles are arranged in close proximity to Ca channels = nanometers
-Very high [Ca] = low affinity vesicles (mM)
‘All-or-nothing’
LDCV = Radial gradients
- Integrate Ca signals from multiple sources; reliant on diffusion
- Low [Ca] = high affinity vesicles
- Diffusion of Ca + transport to the active zone = delay
Microdomains
- More reliable excitation-coupled signals
- Integrate Ca from multiple signals
ie. Auditory system - ensure each electrical signal results in Ca release
Synaptotagmin
Ca sensor in SCV exocytosis (not post-synaptic AMPAR exocytosis)
Interacts with SNARE complex
Contains 2x C2 domains = rigid, oval complexes which possess Ca-binding sites
Ca binds to C2 domain = Ca-dependent phospholipid increased affinity + binding
Exact mechanisms of synaptotagmin is unclear!!!!
- Displace complexin fusion clamp + bind to SNARE complex
- C2 domains insert into the membrane = induction of positive membrane curvature
- Cross-linking the vesicle + PM therefore accelerating fusion
- Likely to play a role in completion of SNARE zippering
DOC2B
Ca sensor in LDCV exocytosis
DO2B = 2x C2 domains - Ca binds, increase affinity for phospholipids
Interacts with Munc13
Promotes Ca-dependent synchronous + asynchronous exocytosis
DOC2B K/O = inhibits asynchronous release (prolonged release whilst the IC [Ca] is still high)
Acts as a priming factor - primes LDCV for fusion (more fusion-competent vesicles; RRP)
Allows for more efficient expansion of the fusion pore - increase catecholamine release via modifying the plasma membrane curvature to stimulate fusion
Munc13
Munc13 K/O = inhibits exocytosis
Interacts with DOC2B
C1, C2 + MUN domain
C2 - involved in Ca-depedent exocytosis
Maybe - unlocks syntaxin from closed conformation bound to Munc18
Isolated MUN domain = restore effects of Munc13 knock-out AND accelerates process from syntaxin-Munc18 –> syntaxin-SNARE complex
Munc18
Munc18 K/O = inhibits exocytosis - unsure why
Binds to syntaxin in closed conformation - unable to form part of SNARE complex
Maybe: recruits to fusion sites where it associates with SNARE motifs and promotes nucleation/zippering
Angiotensin II
Chromaffin cell
AngII receptors couple to multiple G-protein pathways
Results show that activation of multiple types of G-proteins + their pathways by a single modulator acting through a single receptor (ATR I) can produce concentration-dependent, bi-directional regulation of exocytosis!
Low concentration = AngI receptor, Gi/o, inhibit VGCC current = inhibit exocytosis
High concentration = AngI receptor, Gq, increase IC [Ca], increase capacitance = promote exocytosis
-requires Ca release from IC stores
Angiotensin Drugs
BIS/CalC = PKC blocker (catalytic/regulatory domain)
PMA phorbol esters = PKC activator (DAG analogue)
CPA = blocks Ca release from IC stores Caffeine = high concentrations can release Ca from IC stores
Histamine
Chromaffin cell
Gq pathway which engages munc13 to increase stimulus-coupled secretion by recruiting vesicles to the RRP
GPCR is therefore able to regulate exocytosis at the level of the SNARE complex to produce rapid STP of the secretory output
H1 receptor = Gq Potentiates exocytosis (increase capacitance), but decreases Ca
Activation of Gq, but DAG does not activate PKC - it activates Munc13
- Activation by PMA phorbol esters/DAG
- CalC blocks (regulatory domain: PKC + Munc13)
- BIS does not block (catalytic domain of PKC)
- Therefore not PKC dependent
Activation of Munc13 = primes vesicles by regulating Munc18 binding to syntaxin’s closed conformation
Histamine - increase the size of the RRP of vesicles!
Chromaffin GPCR regulation
Angiotensin II - ATR I
Low = Gi/o - decrease exocytosis
High = Gq - increase exocytosis
Activation of multiple types of G-proteins/pathways by a single modulator acting at 1 receptor can produce concentration-dependent bi-directional regulation of exocytosis
Histamine - HI R - Gq
Activation increases the activity of Munc13 (via DAG; PKC-independent) - GPCR is therefore able to regulate exocytosis at the level of the SNARE complex to produce rapid STP of the secretory output
Both act to increase the size of the RRP by shifting vesicles from the reserve pool –> RRP (primed)
Amperometry
Electrochemical detection of catecholamine release in real-time
Dopamine, adrenaline, noradrenaline - oxidised at positive potentials
Carbon fibre electrode charged to positive potential - close to release sites (~5um) - detection of catecholamine release in ‘diffusion spikes’
LDCV - fusion pore + regulation
~200-500nm = larger pores, therefore pore diameter can be regulated
Capable of kiss + run fusion
Amperometry - see ‘foot signals’ = a fraction of the vesicle content is released through the unstable fusion pore before pore closure or collapse
Dynamin regulates fusion events!
Dynamin - normally phosphorylated
High stimulation - calcineurin is activated = deP dynamin, reveals binding sites for accessory proteins
Recruits myosin II = dilates the fusion pore to facilitate peptide release
LINK SCB:
Dynamin = involved in CME (GTPase; PICK1 binding stimulates polymerisation + pinching off the newly forming vesicle)
Myosin = MyoVb involved in post-synaptic exocytosis of AMPARs; Ca binds (sedimentation assay: folded-to-extended), exposes binding domains for RabII = recruit recycling endosomal into the dendritic spine
Kiss + run and full fusion
SCV - mainly full fusion
LDCV - both!
Kiss + run fusion = increases with age
Age - the kinetics of full fusion and kiss + fun fusion differ with age - alter differentially - implies that there are different regulatory mechanisms
SCV fusion
~50nm = very small therefore always expand to full fusion
Small pore - due to the physics of membrane curvature, most are full fusion
Parkinson’s Disease
Treatment = L-DOPA - enhanced DAergic neurotransmission via increased pool of vesicular dopamine (RRP) = increased release events seen on amperometry
VMAT2 = drug target for enhanced DAergic transmission - either increasing VMAT2 or enhancing current protein
VMAT2 dysfunction has been identified in PD brains!
Reserpine PD-model = bind to VMAT2 and reversible inhibit its’ function
Microamperometry
Detection of single secretory vesicles in individual cells
Positively charged carbon fibre except 5um tip!
Close proximity required to pick up diffusion of transmitters when it is released via exocytosis
Tracer of release events = higher spike, larger area under diffusion curves, more catecholamine released
Spontaneously hypertensive rats
RVLM = rostral ventrolateral medulla - A2 + C1 region
Electron microscopy = larger size of vesicles in C1
Microamperometry = larger quanta of catecholamines released per exocytic event in C1 neurones
Electrophysiology = low AngII (Gi/o; decrease IC [Ca]) - less inhibition of Ca influx therefore less inhibition of exocytosis
Leads to increased sympathetic activity = increase blood pressure in SHR!
Noradrenergic vesicles
NAergic neurones in A1, A2 + OSC
Noradrenaline in peripheral noradrenergic nerves is released exclusively from LDCVs by an exocytotic mechanism
TTX (VGSC blocker) eliminated majority of exocytic events - mainly, but not entirely, AP driven
SCV, DCV, LDCV
Main population = SCV + DCV
Astrocyte-neurone lactate-shuttle hypothesis
High neuronal activity = increased K+ uptake (depolarise) + increased glutamate (EAAT1/2)
Trigger glyogenolysis (glycogen –> glucose)
Glucose –> pyruvate –> lactate
Lactate released via MCT1 (astrocyte)
Lactate taken up via MCT2 (neurones)
Oxidised to pyruvate = enter Krebs cycle for ATP!
EC [lactate] increases with stimulation
***MCT used H+ as a co-substrate!
Astrocytes regulating post-synaptic AMPARs
Hypothalamus
Adrenaline binds to alpha-1 receptors on astrocytes - release ATP - bind to P2X7 receptors on glutamatergic cells, cause an increase in AMPAR insertion
Gliotransmitter contributes directly to the regulation of postsynaptic efficacy at glutamatergic synapses in the CNS
CONTROVERSIAL = Astrocytic release machinery
Glutamate
Exo = BoTx/tetanus application decreased Ca-dependent glutamate release
or
Activation of P2X receptor, increase [Ca], activate VRAC receptor, release glutamate
ATP
Hemichannel blocker inhibits ATP release in astrocytic hippocampal slice
TIRF = observation of exocytosis of secretory lysosomes following activation
Astrocytic vs Neuronal exocytic machinery
SNAP-25 (neurone) + SNAP-23 (astrocyte)
Synaptotagmin-1 (neurone) + synaptotagmin 4, 7 or 11 (astrocyte)
Synaptobrevin-2 (neurone) + synaptobrevin-3 (astrocyte)
Slower kinetics in astrocytes - involves GPCR signalling + IP3-mediated Ca release from IC stores!!!
(Neuronal = VGCCs)
CONTROVERSIAL: Synaptotagmin
Synaptotagmin in astrocytes
Synaptotagmin 4 K/O = reduced SNARE-mediated exocytosis of glutamate
BUT
mRNA expression study (qRT-PCR) = astrocytes express little synaptotagmin 4 and lots of synaptotagmin 11
BUT - mRNA expression does not equal protein expression
Maybe look at Western blots?
Astrocytic syncytium
Astrocyte connected cell network via gap junctions - each astrocyte contributes a hemichannel (6x connexins; gap junction = 2 hemichannels)
IC signalling network - propagate Ca waves throughout the network
Paracrine via ATP release (exocytic??) - acts via gap junctions
Highly controversial field
Modulation of fast synaptic transmission
Astrocytes in the cortex can modulate the inhibitory activity of neurones in the neocortex
Astrocytes release ATP, bind to P2X receptors, cause a decrease in IPSP in pyramidal neurones
Down-regulation of inhibitory synaptic currents (IPSPs) in the neocortex due to astrocytic ATP release binding to P2XRs
Astrocytic involvement in learning + memory
Lactate released via MCT1 from astrocytes - neurones take it up via MCT2 - enter Krebs cycle for ATP
In vivo:
Disrupt MCT1 = causes amnesia; rescued by EC lactate
Disrupt MCT2 = causes amnesia; cannot be rescued by EC lactate
Lactate shuffle = important for L+M - long-term memory formation and maintenance of LTP (disrupt = amnesia)
Disrupting the expression of astrocytic MCT1 = causes amnesia + LTP impairment
Astrocytes + aging
Age-related disease in astrocytic Ca signalling - decrease in exocytosis of gliotransmitters, particularly ATP
Cortical astrocytes - decrease of astrocytic modulation of synaptic transmission (ie. modulating fast synaptic transmission) could contribute to age-related impairment synaptic plasticity and cognitive impairment
Astrocytes + breathing
Ventral medulla surface (VMS) in brainstem
Astrocytes = chemosensitive; blood acidosis = activate paracrine ATP signalling in astrocytes
(ATP binds P2X/YRs, IC [Ca], release ATP)
ATP activates phrenic nerve/central inspiratory drive = activate diaphragm muscles (major respiratory muscle) = increase breathing!
Experimentally:
In vivo artificially anaesthetised + hyperventilated rat
Optogenetically stimulate astrocytes - activates phrenic nerve activity = increase breathing
Astrocytes + arousal
Astrocytes release lactate (MCT1)
Lactate shuffle not involved!
Bind to Gs-coupled receptors on NAergic neurones in the locus coeruleus - release noradrenaline
= NAergic input into the cortex - arousal, alertness, motivation, sleep/wake cycle
Micro-injections of lactate into LC = arousal in anaesthetised rats
- Increased arterial blood pressure
- Power changes in the cortex (sleeping state –> awake state)
Lactate = acting as a gliotransmitter to control arousal!
Astrocytes + hypertension
Brainstem hypoxia contributes to the development of hypertension in SHR
Hypoxia = lower O2 in brainstem
Astrocytes = activated (low pH) - increased paracrine ATP signalling (Ca waves)
Increased release of lactate
Act as a gliotransmitter to increase central inspiratory drive (via increasing phrenic nerve activity) to increase breathing
Increased sympathetic drive
Increased exocytosis in RVLM:
-Larger LDCV (EM) + catecholamine release (microamperometry)
= larger NA release = increased BP!!!
Alternative model to ‘Docked + Primed’
SNAREs do not form a complex/zipper before the Ca signal arrives
Ca signal triggers cross-linking of the vesicle + PM = close contact! Close proximity allows for rapid binding of vSNAREs + tSNAREs
Nucleation triggered = SNAREs zipper + fusion/exocytosis occurs
Entire control of fusion is up-stream of SNARE nucleation - no pausing of the highly exergonic process (the mechanism of complexin arresting SNARE zippering in the middle is difficult to understand because SNARE assembly precedes along a steep downhill energy gradient; strongly exergonic reaction)
SUPPORT:
Simple model - therefore highly conserved throughout evolution
Mutated SNAREs affect zippering due to increases in ‘misfiring’ - assembly without fusion
Explains why sucrose triggers Ca-independent exocytosis of RRP
- Sucrose causes water efflux = negative pressure in the cell
- Negative pressure draws docked vesicles closer to the plasma membrane = triggers SNARE firing
C2 domain
Ca binds, causes an “electrostatic switch” - shield negatively charged residues on the phospholipid binding site, therefore facilitating binding to negatively charged phospholipids
