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Pharmacology of Ion Channels > Dr Anja Teschemacher > Flashcards

Dr Anja Teschemacher Flashcards

1
Q

Connexins

A 
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

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2
Q

Pannexins

A

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

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3
Q

Connexins + pannexins

A

Do not show high sequence homology!!

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4
Q

Membrane transporters - MCT

A

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

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5
Q

Membrane transporters - glutamate transporters

A 
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

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6
Q

Heteroexchange

A

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)

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7
Q

Cocaine/amphetamines

A

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

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8
Q

Cross-talk between transporter regulation mechanisms

A

GAT1 = GABA transporter 1
Co-substrate = Na + Cl
Uses energy from the dissipation of a Na+ gradient

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9
Q

SCV

A

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

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10
Q

LDCV

A

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

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11
Q

Lipid bilayer fusion

A

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

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12
Q

SNARE proteins

A

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

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13
Q

Tetanus

A

Wound infection

Cleave vSNAREs of motor inhibitory interneurones

Progressive muscle spasms (due to loss of inhibition)

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14
Q

Botulinum toxin (Botox)

A

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

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15
Q

TIRF

A

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

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16
Q

Complexin

A

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)

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17
Q

Flash photolysis

A

Caged Ca - EGTA-photosensitive cage
Whole-cell patch clamp
EGTA = calcium cheater - photosensitive

UV flash = uniform [Ca] elevation, measure exocytic events

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18
Q

Membrane capacitance studies

A

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!

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19
Q

Calcium requirements of SCV

A

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!

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20
Q

Domains - SCV + LDCV

A

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
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21
Q

Synaptotagmin

A

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
22
Q

DOC2B

A

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

23
Q

Munc13

A

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

24
Q

Munc18

A

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

25
Q

Angiotensin II

A

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

26
Q

Angiotensin Drugs

A

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
27
Q

Histamine

A

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!

28
Q

Chromaffin GPCR regulation

A

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)

29
Q

Amperometry

A

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’

30
Q

LDCV - fusion pore + regulation

A

~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

31
Q

Kiss + run and full fusion

A

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

32
Q

SCV fusion

A

~50nm = very small therefore always expand to full fusion

Small pore - due to the physics of membrane curvature, most are full fusion

33
Q

Parkinson’s Disease

A

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

34
Q

Microamperometry

A

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

35
Q

Spontaneously hypertensive rats

A

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!

36
Q

Noradrenergic vesicles

A

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

37
Q

Astrocyte-neurone lactate-shuttle hypothesis

A

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!

38
Q

Astrocytes regulating post-synaptic AMPARs

A

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

39
Q

CONTROVERSIAL = Astrocytic release machinery

A

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

40
Q

Astrocytic vs Neuronal exocytic machinery

A

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)

41
Q

CONTROVERSIAL: Synaptotagmin

A

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?

42
Q

Astrocytic syncytium

A

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

43
Q

Modulation of fast synaptic transmission

A

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

44
Q

Astrocytic involvement in learning + memory

A

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

45
Q

Astrocytes + aging

A

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

46
Q

Astrocytes + breathing

A

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

47
Q

Astrocytes + arousal

A

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!

48
Q

Astrocytes + hypertension

A

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!!!

49
Q

Alternative model to ‘Docked + Primed’

A

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
50
Q

C2 domain

A

Ca binds, causes an “electrostatic switch” - shield negatively charged residues on the phospholipid binding site, therefore facilitating binding to negatively charged phospholipids

Pharmacology of Ion Channels (9 decks)

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