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BIOL 302 - CELL SIGNALLING, TRANSPORT + DISEASE > The Calcium Signalling Toolkit > Flashcards

The Calcium Signalling Toolkit Flashcards

1
Q

What is the calcium signalling toolkit?

A

Ca2+ = a UBIQUITOUS component of signalling (a second messenger)

= important in many pathways:
(e.g. exocytosis, contraction, metabolism, gene transcription, fertilisation, proliferation, hypertrophy) = they are very highly conserved

= very accurate and specific with high fidelity

Calcium signalling toolkit
= molecular machinery cells use to detect and respond to chances in Ca+ concentration + generate specific physiological responses
= involves:
= calcium channels
= calcium sensors
= calcium buffers
= calcium pumps and exchangers
= calcium-dependent enzymes

= dysregulation can lead to variety of diseases

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2
Q

What is an example of a calcium response in Pancreatic Acinar Cells?

A

CCK (cholecystokinin)
= stimulates α-amylase secretion

= produced in response to peptides, amino acids in the duodenum

= CCK causes increase in cytosolic calcium concentration = which triggers the machinery responsible for secreting α-amylase

= production of α-amylase = enters duct = into gut = digestion

= when CCK binds to receptor on surface of acinar cell = triggers series of intracellular signalling events = leads to increase in concentration of Ca2+ in cytosol

= dysregulation of the calcium signalling pathway can lead to disorders such as pancreatitis

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3
Q

How to measure cytosolic Ca2+ concentrations?

A
  1. Photoproteins (e.g. Aequorin)
    = apopaequorin (inactive) - binds to coelenterazine = aequorin (active)
    = aequorin in contact with Ca2+ = produces blue light
    (proportionate)
  2. Fluorescent Ca2+-sensitive indicators (e.g. Fura-2)
    = selectively bind to calcium ions to produce a fluorescent signal in response to changes in calcium concentration
    = upon binding = conformational change = change in fluorescent signal emmited by fluorophore
    = can monitor in real time through fluorescence microscope or plate reader
  3. GECIs / FRET = Genetically encoded Fluorescence Resonance Energy Transfer - based Ca2+ sensors (e.g. Ca2+ cameleons)
    = 2 fluorescent protein are genetically fused to a calcium-binding protein (e.g. calmodulin)
    = first fluorescent protein acts as donor fluorophore
    = second fluorescent protein act as acceptor fluorophore
    = when calcium binds = conformational change = changes distance between donor and acceptor
    = changes FRET ratio = can detect changes in intracellular calcium concentration
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4
Q

What type of information does measuring calcium levels give?

A

Quantitative and temporal information
= e.g. aequorin transformed yeast

Quantitative, temporal AND spatial information
= e.g. Fura-2-loaded sea squirt eggs

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5
Q

What are some mechanisms for generating increases in cytosolic calcium conentration?

A

= ON mechanisms

= many across animals, plants, budding yeast

= range of channels in plasma membrane / endomembrane
(either Ca2+-only permeable OR cation channels that are permeable to Ca2+)

Ca2+ entry
= influx across the plasma membrane
(down concentration gradient into cytosol)

Ca2+ release
= from intracellular stores

(OFF mechanisms = Ca2+ buffers, mitochondria, Na+/Ca2+ exchanger, Ca2+ pumps)

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6
Q

What are some examples of plasma membrane calcium channels? (Ca2+ entry)

A
  1. VOCs (voltage operated calcium channels)
    = gated by membrane potential of cell (depolarisaton / hyperpolarisation)
    = CaV = voltage-gated Ca2+ channel
    = TRP = transient receptor potential channel
  2. ROCs (receptor operated calcium channels)
    = bind a ligand = conformational change = gating
    = iGluR = ionotropic glutamate receptor
    = P2XR = ionotropic ATP receptor
  3. SOCs (store operated calcium channels)
    = gated by calcium levels in stores
    = allows influx to refill stores
    = CRAC = orai
  4. SMOCs (second messenger operated calcium channels)
    = 2nd messengers = cyclic nucleotides = changes = causes differences in calcium
    = CNG, cyclic nucleotide-gated channel
  5. Ca2+ permeable mechanosensitive channels
    = physical change (e.g. stretching of PM - allows influx)
    = PIEZO and CSC/OSCA
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7
Q

What are examples of Ca2+ release?

A

= Phospholipase C-inositol (1,4,5) triphosphate (PLC-IP3) based pathway

= ON mechanism (Ca2+ release from endomembrane stores)

= ligand binds to GPCR on cell surface
= activates PLC
= cleaves PIP2 into DAG and IP3
= DAG = downstream PKC phosphorylation
= IP3 binds to IP3R on membrane of ER
= triggers opening of calcium channel in ER membrane
= leads to release of calcium ions in cytosol

= released calcium can bind and activate other calcium-binding proteins
(e.g. calmodulin, troponin C)
= initiates downstream signalling cascades (PKC - phosphorylation)
= rise in cytosolic calcium concentration can also activate plasma membrane calcium channels = further influx into cell
= CICR (calcium-induced calcium release - feedforward system)

(Activation of PKC)
= glycogen metabolism (glycogen synthase)
= gene expression (transcription factors)

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8
Q

What is the structure of GPC(Rs)?

A

= G proteins + G protein-coupled receptors

= superfamily with conserved motifs
= 7 TMS α-helices
= 4 extracelullar + 4 cytosolic domains = transduce signal from receptor to G protein

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9
Q

What are the 3 principal groups of G-protein-coupled receptor?

A

Family A
= responds to:
= biological amines
= light or odorants
= peptides or chemokines
= purines
= lipids

Family B
= responds to peptides
= large extracellular domain attached to 1st TMS = ligand binding

Family C
= venus flytrap domains (capturing / receiving extracellular stimulus
= responds to:
= biological amines
= glutamate
= Ca2+
= large extracellular domain
= frequently form dimers

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10
Q

How do G proteins act as molecular switches / timers?

A

Heterotrimeric G protein
= alpha, beta and gamma subunits
= has guanyl nucleotide binding site
(can bind to GDP/GTP)

Molecular switches
= GDP bound to Gα = OFF
= GTP bound to Gα = ON

Molecular tiers
= High GTPase activity = BRIEF activation
= Low GTPase activity = PROLONGED activation

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11
Q

How does GPCR and effector protein activation occur?

A
  1. Binding of hormone
    = induces conformational change in receptor
  2. Activated receptor binds to Gα subunit
  3. Activated receptor causes conformational change in Gα
    = triggering dissociation of GDP
  4. Binding of GTP to Gα
    = triggers dissociation of Gα both from receptor + Gβγ
  5. Hormone dissociates from receptor Gα
    = binds to effector, activating it
  6. Hydrolysis of GTP to GDP
    = causes Gα to dissociate from effector
    = reassociate with Gβγ

= can be measured using FRET
(activation through cAMP - extracellular ligand = measure fluorescence when cAMP is added)

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12
Q

What are some G protein responses?

A

Split into Gα and Gβγ responses

E.g. of Gβγ and Gα response
= PLC (phosphalipase C) = DAG + IP3 = Ca2+

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13
Q

What is Phospholipase C (PLC)?

A

= 16 members of Phospholipase C family

= Classical PI-PLCs = conserved structure
(beta, gamma, delta, epsilon, zeta, eta)

= Atypical PI-PLCs
(PLC-XD)

PLC = hydrolysed to DAG and IP3 (second messengers)

(see IP3 pathway on other card)

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14
Q

How does Ca2+ release from intracellular stores occur?

A

e.g. from ER/ SR/ lysosome-related organelle

= efflux of Ca2+ from endomembrane stores = ON mechanism

= requires additional Ca2+ mobilising molecules:

cADPR
= cyclic ADP ribose
= requires enzyme ADP ribosyl cyclase to turn NAD+ to cADPR
= ER / ryanodine receptor Ca2+ channel

NAADP
= nicotinic acid adenine dinucleotide phosphate
= requires enzyme ADP ribosyl cyclase to turn NADP to NAADP
= lysosome-like organelle

also S1P = sphingosine-1-phosphate
= mobilised Ca2+ release from endomembrane stores

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15
Q

How does S1P impact calcium signalling?

A

S1P bind to S1P receptors
(which are GPCRs that activate signalling pathways)

S1PR activation leads to release of calcium from intracellular stores into cytoplasm
= which then activates downstream effectors
(e.g. calmodulin / protein kinase C)

Also has intracellular targets = e.g. IP3R
= binds to and modulates activity = impacts intracellular calcium levels

= requires SK1 enzyme = sphingosine kinase

= site of action unknown

evidence:
= GPCR-independent increase in cytosolic calcium concentration in pertussis toxin-treated cells (inhibits GPCR signalling)
= was stimulated by S1P

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16
Q

How are Ca2+ stores replenished?

A

= through STIM proteins (stromal interaction molecule)

= process of refilling calcium stores
= called CCE (capacitative calcium entry) or SOCE (store-operated calcium entry)

= STIM protein on ER membrane

= when ER stores depleted = STIM proteins sense decrease = undergo conformational change = oligomerise and form clusters near plasma membrane

= then interact wit Orai proteins (calcium channels in PM)

= opening of Orai channels = entry of extracellular calcium into cytoplasm

= refills calcium stores in ER
(highly regulated to ensure proper calcium homeostasis)

= CAD domains
(interact with store-operated channels - Orai1)

= EBI
(prevents movement of STIM proteins by microtubule interaction)

STIM 1 = inhibits plasma membrane voltage-gated Ca2+ channels (CaV1.2)
= diseases with mutations in channel = prevent inhibition by STIM protein = too much Ca2+ let in
(e.g. Timothy and Brugada syndromes - cardiac arrhythmias = CaV1.2 gene CACNA1C targeted by drugs: verapamil, dilitiazem)

EXTRA READING
= STIM proteins also interact with other ion channels and transporters to regulate calcium homeostasis
= e.g. TRPC channels and the Na+/Ca2+ exchanger

17
Q

What are some OFF mechanisms?

A

= return cytosolic calcium concentrations to pre-stimulation levels

= reset Ca2+ signalling

= stop toxicity

= can contribute to specificity

= Ca2+ buffers
(localise Ca2+ signals = can’t spread)

= Ca2+ efflux pathways
(decreases Ca2+ concentrations)

18
Q

What are examples of Ca2+ efflux pathways?

A

= removal from cytosol out of cell / into store

PMCA
= Plasma membrane Ca2+-ATPase
= pumps out of cell across PM

NCX
= Plasma membrane Na+/Ca2+ exchanger
= exchanges Na+ for Ca2+

e.g. SERCA
= ER and SR located Ca2+-ATPases
= move into ER as a store

Mitochondrial uniporter
= acts as store

19
Q

What are examples of Ca2+ sensors

A

= downstream of cytosolic calcium concentration elevations

= Ca2+ sensitive proteins, have EF hand domains = bind Ca2+

e.g.
= CaM - Calmodulin

= TnC - Troponin C
= NCS proteins - Neuronal Ca2+ Sensors
= CaBPs - Ca2+-binding proteins
= CIB1 - Calciu and integrin binding protein 1
= S100 proteins (divergent functions)
= Annexins
= Synaptotagmins
(plus more)

20
Q

What is Calmodulin (CaM)?

A

structure = 4 EF hands = binds 4 Ca2+

= acts a molecular switch:

Increased calcium ions in cytosol
= calcium binds bind to CaM
= conformational change in protein
= enables interaction with target proteins = CaMBDs
= can modulate activity of target protein

= can bind up to 4 calcium ions = highly sensitive sensor
= binding of each ion causes structural change = increases affinity for additional calcium ions
= (called calcium-induced calcium release / calcium cooperativity)
= enables small changes to be detected and respond with strong signal

EXTRA READING
= also acts as calcium buffer
= binding excess calcium ions = preventing them from disrupting cellular function
= needs to be in balance with buffer and sensor

21
Q

What are some examples of Ca2+ effectors

A

= downstream of cytosolic calcium concentration elevations

e.g.
= Ca2+/CaM-dependent protein kinases (CaMKs)

= Ca2+-sensitive K+ channels
= Ca2+-sensitive Cl- channels
= Calcineurin (CaN) - protein phosphatase 2B (PP2B)
= Ca2+-sensitive Ras-GTPase-Activating Proteins (e.g. CAPRI, RASAL)
= Phosphorylase kinase
= MLCK - Myosin light chain kinase

22
Q

What are CaMKs?

A

= calmodulin kinases
(serine/threonine protein kinases activated by binding to calcium-bound calmodulin)

= several CaMK isoforms
(e.g. CaMKI, CaMKII, CaMKIV and CaMKK = have unique tissue distribution and functions)

When calcium binds to calmodulin
= Ca2+-calmodulin complex interacts with CaMKs
= activation (through autophosphorylation - kinase phosphorylates itself = increased kinase activity)

Activated CaMKs
= phosphorylate variety of downstream targets
= can modulate their activity

BUT for CaMKI / CaMKIV
= binding to complex induces conformational change
= requires phosphorylation by CaMKK (not autophosphorylation) for activation

EXTRA READING
= e.g. CaMKII = involved in regulation of synaptic plasticity
= increased kinase activity = leads to phosphorylation of AMPA receptors (ion channels that mediate majority of fast excitatory neurotransmission in brain)
=e.g. CamKIV = involved in regulation of gene expression in response to calcium signalling
= when activated can enter nucleus and phosphorylate transcription factors = changes in gene expression / cellular function

BIOL 302 - CELL SIGNALLING, TRANSPORT + DISEASE (11 decks)

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