Calcium signalling toolkit 2

Question 1

Phospholipase C

Answer 1

16 member family
Can be activated by heterotrimeric G protein
All hydrolyse PIP2 to produce diacylglycerol (DAG) which remains in the membrane or IP3 (soluble)

Question 2

IP3 signalling

Answer 2

Active PLC hydrolyses PIP2
IP3 diffuses through membrane to IP3R (gated calcium channel) in the ER
Binding allows Ca2+ efflux from intracellular stores
Ca2+ release makes receptor more sensitive to IP3 so more release (calcium-induced calcium release)
At high Ca2+, IP3 binding is inhibited

Question 3

DAG

Answer 3

Membrane DAG binds to DAG binding site on PKC (protein kinase C) leading to activation
PKC phosphorylates downstream targets (glycogen synthase, TFs)

Question 4

cADPR

Answer 4

Cyclic ADP ribose
Release of Ca2+ from ER through ryanodine receptor (channel)
Acts on SERCA pumps which fill ER with calcium to cause release

Question 5

NAADP

Answer 5

Nicotinic acid adenine dinucleotide phosphate
Release of calcium from lysosome-related organelles
Activates 2-pore calcium channels (TPC1/2)

Question 6

S1P

Answer 6

Sphingosine-1-phosphate
Produced by sphingosine kinase in the cell and exported
Binds S1PR (GPCR) to cause calcium increase through IP3-PLC pathway

Question 7

Stromal interaction molecule proteins

Answer 7

STIM1 protein in the ER
Lumenal domain is calcium sensor (EF hands)
CAD region in cytosol interacts with store-operated channels (Ori1)
When ER is full, Ca2+ is bound to EF hands. STIM1 binds to microtubule via EB1 (fixed in place)

Question 8

Intracellular store refilling

Answer 8

Ca2+ depleted in ER, dissociate from EF hand.
STIM proteins oligomerise, weakening EB1 binding.
Relocate to area of ER close to plasma membrane.
CAD domains bind and trigger opening of store-operated channels. Influx of cytosolic calcium is then pumped into stores

Question 9

CaV1.2 channels

Answer 9

STIM1 inhibits plasma membrane voltage-gated Ca2+ channels
Prevent constant influx of Ca2+
Mutations in Cav1.2 gene CACNA1C causes Timothy and Brugada syndromes
Verpamil drug targets and blocks Cav1.2

Question 10

Ca2+ buffers

Answer 10

Regulate Ca2+ concentration
Can prevent spread of calcium ions and signal (maintains signal in discrete region)
Act at peak of calcium transient

Question 11

Ca2+ efflux pathways

Answer 11

Plasma membrane Ca2+ ATPase (PMCA)
Plasma membrane Na+/Ca2+ exchanger (NCX)
ER/SR located Ca2+ ATPases (SERCA)
Mitochondrial uniporter

Act at wide range in calcium transient

Question 12

Calmodulin (CaM)

Answer 12

4 EF hands (binding domain between E and F helices)
Calcium binds EF hands when there is an increase in cytosolic calcium
CaM conformational change allowing interaction with and activation of downstream effectors

Question 13

CaMKs

Answer 13

Ca2+/calmodulin-dependent protein kinases
Downstream effector
Both CaMKs and CaMK kinases are activated by calmodulin/calcium

Question 14

CaMK activation

Answer 14

Ca/CaM binds to binding domain on CaMKK causing conformational change and activation.
Also activates CaMK
CaMKK phosphorylates autoinhibitory site on CaMK for full activation
CaMK phosphorylates downstream effectors

Question 15

Annexins

Answer 15

Phospholipid binding Ca2+ sensors
Annexin A7 (Synexin) causes aggregation of chromaffin granules in the adrenal glands in the presence of free calcium
Form a complex with S100 proteins and can regulate CFTR channels
Mutation in Annexin A11 associated with sarcoidosis