PSC2002/L22 Dynamics of cAMP Signalling Flashcards
How are cAMP dynamics measured in living cells? (3)
Genetically-encoded fluorescent sensors
Using cAMP binding domains (BD) from PKA or EPAC attached to fluorescent proteins
Sensors expressed in cells using plasmids
What are the 2 main approaches to measuring cAMP dynamics in living cells?
FRET
Intensity
Describe the FRET method of measuring cAMP dynamics in living cells.
Uses cAMP BD - has 2 different fluorescent proteins (CFP/YFP) attached that undergo FRET (decreases when cAMP rises)
Describe the intensity method of measuring cAMP dynamics in living cells.
cAMP BD + fluorescent protein tag
Rise in cAMP increases fluorescent intensity
Describe the Zaccolo and Pozzan experiment, 2002.
Excited cell and activate with light
Separates proteins from each other
cAMP levels increase - FRET decreases
Why do 2 different GPCR cAMP agonists cause different functional effects in heart cells? (2)
Different GPCR subtypes & signalling pathways
Differential cAMP compartmentalisation
Different effector proteins
What is phospholamban?
Negative regulator of SERCA
Until it gets phosphorylated
What controls spatial localisation of cAMP?
Phosphodiesterases localised to different regions
Where is activation of B-adrenergic receptors localised to?
T-tubules & SR
Give 3 examples of proteins involved in excitation-contraction coupling.
Dihydropyridine receptors (LTCCs)
Phospholamban
Ryanodine 2 receptors
What can lead to phosphorylation of enzymes involved in metabolism and transcriptional factors?
Activation of prostanoid receptors
What is the key role of PDEs?
Regulating spatial patterns of cAMP signals and functional responses from cardiac cells
Describe phenotypic remodelling.
Specific to each receptor
Dependent on spatial changes in cAMP & localisation of downstream signalling molecules (cAMP signalsome)
What is the result of increased force of contraction induced by B-adrenergic receptors during exercise?
PKA-dependent phosphorylation of key Ca2+ signalling components
L-type calcium channels
Phospholamban
Ryanodine type 2 receptors
What effect does phenotypic remodelling have on the heart during exercise? (3)
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
What is the effect of phenotypic remodelling by PKA phosphorylation on LTCC?
Activity increases
What is required for increase in LTCC activity by cAMP/PKA?
AKAP
How does PKA increase LTCC activity? (2)
PKA phosphorylation increases open state probability (Po) of individual LTCCs in sarcolemma
PKA recruits more LTCCs to sarcolemma (increase N)
What was the result of beta-adrenergic receptor stimulation of isolated mouse cardiomyocytes?
Increased number of LTCCs at sarcolemma
Formation of ‘super clusters’ of LTCCs
How were LTCCs ‘visualised’ during B-adrenergic receptor stimulation of isolated mouse cardiomyocytes?
By attaching a GFP-tag to LTCCs and expressing them in cardiac cells
How do ‘super clusters’ of LTCCs form?
Superclusters of LTCCs formed by interaction of their C-terminal domains, only after PKA-phosphorylation of LTCC
Describe the role of phospholamba (PLB). (2)
PKA phosphorylation
Increases in Ca2+ causes dissociation of PLB from SERCA
Why do cAMP and calcium signalling interact?
To maximise contractile response from cardiac cells
How is spatial cAMP signalling disrupted in CF airway cells? (3)
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
