PSC2002/L22 Dynamics of cAMP Signalling

Question 1

How are cAMP dynamics measured in living cells? (3)

Answer 1

Genetically-encoded fluorescent sensors
Using cAMP binding domains (BD) from PKA or EPAC attached to fluorescent proteins
Sensors expressed in cells using plasmids

Question 2

What are the 2 main approaches to measuring cAMP dynamics in living cells?

Answer 2

FRET
Intensity

Question 3

Describe the FRET method of measuring cAMP dynamics in living cells.

Answer 3

Uses cAMP BD - has 2 different fluorescent proteins (CFP/YFP) attached that undergo FRET (decreases when cAMP rises)

Question 4

Describe the intensity method of measuring cAMP dynamics in living cells.

Answer 4

cAMP BD + fluorescent protein tag
Rise in cAMP increases fluorescent intensity

Question 5

Describe the Zaccolo and Pozzan experiment, 2002.

Answer 5

Excited cell and activate with light
Separates proteins from each other
cAMP levels increase - FRET decreases

Question 6

Why do 2 different GPCR cAMP agonists cause different functional effects in heart cells? (2)

Answer 6

Different GPCR subtypes & signalling pathways
Differential cAMP compartmentalisation
Different effector proteins

Question 7

What is phospholamban?

Answer 7

Negative regulator of SERCA
Until it gets phosphorylated

Question 8

What controls spatial localisation of cAMP?

Answer 8

Phosphodiesterases localised to different regions

Question 9

Where is activation of B-adrenergic receptors localised to?

Answer 9

T-tubules & SR

Question 10

Give 3 examples of proteins involved in excitation-contraction coupling.

Answer 10

Dihydropyridine receptors (LTCCs)
Phospholamban
Ryanodine 2 receptors

Question 11

What can lead to phosphorylation of enzymes involved in metabolism and transcriptional factors?

Answer 11

Activation of prostanoid receptors

Question 12

What is the key role of PDEs?

Answer 12

Regulating spatial patterns of cAMP signals and functional responses from cardiac cells

Question 13

Describe phenotypic remodelling.

Answer 13

Specific to each receptor
Dependent on spatial changes in cAMP & localisation of downstream signalling molecules (cAMP signalsome)

Question 14

What is the result of increased force of contraction induced by B-adrenergic receptors during exercise?

Answer 14

PKA-dependent phosphorylation of key Ca2+ signalling components
L-type calcium channels
Phospholamban
Ryanodine type 2 receptors

Question 15

What effect does phenotypic remodelling have on the heart during exercise? (3)

Answer 15

Generates larger Ca2+ signals, increased trigger Ca2+ via PTCCs, increased Ca2+ store release via RyR2 (inotropy)
Removal of Ca2+ quicker into stores during diastole to enable faster relaxation (lustropy)
Reload Ca2+ stores better to help increase inotropy

Question 16

What is the effect of phenotypic remodelling by PKA phosphorylation on LTCC?

Answer 16

Activity increases

Question 17

What is required for increase in LTCC activity by cAMP/PKA?

Answer 17

AKAP

Question 18

How does PKA increase LTCC activity? (2)

Answer 18

PKA phosphorylation increases open state probability (Po) of individual LTCCs in sarcolemma
PKA recruits more LTCCs to sarcolemma (increase N)

Question 19

What was the result of beta-adrenergic receptor stimulation of isolated mouse cardiomyocytes?

Answer 19

Increased number of LTCCs at sarcolemma
Formation of ‘super clusters’ of LTCCs

Question 20

How were LTCCs ‘visualised’ during B-adrenergic receptor stimulation of isolated mouse cardiomyocytes?

Answer 20

By attaching a GFP-tag to LTCCs and expressing them in cardiac cells

Question 21

How do ‘super clusters’ of LTCCs form|?

Answer 21

Superclusters of LTCCs formed by interaction of their C-terminal domains, only after PKA-phosphorylation of LTCC

Question 22

Describe the role of phospholamba (PLB). (2)

Answer 22

PKA phosphorylation
Increases in Ca2+ causes dissociation of PLB from SERCA

Question 23

Why do cAMP and calcium signalling interact?

Answer 23

To maximise contractile response from cardiac cells

Question 24

How is spatial cAMP signalling disrupted in CF airway cells? (3)

Answer 24

Defective CFTR activated by PKA via cAMP signalling
Altered CFTR function & changes in expression of ACs and PDEs mean dysregulated cAMP levels and activation of different downstream signalling pathways
Reduced cAMP response