lecture 22

Question 1

how do we measure cAMP dynamics in living cells?

Answer 1

-use genetically encoded florescent tenses to study spatiotemporal cAMP dynamics
-utilise cAMP binding domains from PKA or EPAC attached to fluorescent proteins

Question 2

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

Answer 2

1. FRET
2. intensity

Question 3

what is the FRET approach?

Answer 3

-uses cAMP binding domain with 2 different flourescent proteins (CFP/YFP) attached that undergo FRET
-FRET decreases when cAMP rises

Question 4

what is intensity approach?

Answer 4

-cAMP binding domain and flourescent protein
-rise in cAMP increases flourescent intensity

Question 5

what do 2 different GPCR cAMP agonists cause different functional effects in heart cells?

Answer 5

-beta adrenergic and PGE receptors
-beta AR activated - increases contractility
-PGER activation = no effect on contractility

Question 6

what happens with beta adrenergic receptors?

Answer 6

-elevation in cAMP localised to T tubules and SR
-causes phosphorylation of key proteins involved in excitation coupling like LTCCs, SERCA regulator and RyR2

Question 6

what to prostanoid receptors lead to?

Answer 6

-phosphorylation of enzymes involved in metabolism and transcription factors
-involved in long term changes in cell

Question 7

what is found when PDEs are inhibited?

Answer 7

-selective PKA phosphoryaltion
-less regulation of spatial patterns in cAMP signalling
-less functional response from cardiac cells

Question 8

what is found about phenotypic remodelling?

Answer 8

-specific to each receptor
-dependent on spatial changes in cAMP
-dependent on localisation of downstream signalling molecules - the signalsome

Question 9

what effects does phenotypic remodelling of cAMP/PKA pathway have?

Answer 9

-increased force of contraction induced by beta-adrenergic stimulation leads to PKA dependent phosphorylation of Ca2+ signalling components

Question 10

what signalling components are phosphorylated?

Answer 10

1. L type calcium channels
2. phospholamban, SERCA pump regulator
3. ryanodine type 2 receptors

Question 11

what does the phosphorylation allow heart cells to do?

Answer 11

-generate larger Ca2+ signals = increased trigger calcium via LTCCs = increased Ca2+ release via RyR2
-remove ca2+ quicker into stores during diastole to enable quicker relaxation (lustropy)
-reload Ca2+ stores better to help increase inotropy

Question 12

how does phenotypic remodelling by PKA phosphorylation increase LTCC activity?

Answer 12

-LTCC activate in isolated cell by the change in voltage
-set membrane potential to a voltage which opens all calcium channels
-forksolin added and calcium is flowing into cell so current is negative

Question 13

what is needed for an increase in LTCC activity?

Answer 13

AKAP-79

Question 14

what happens when you prevent AKAP2 cardiac cells?

Answer 14

reduced ability to stimulate calcium channels

Question 15

how does phenotypic remodelling by cAMP/PKA pathway affect the SERCA pump?

Answer 15

-phosphorylation of PKA and increase in Ca2+ = dissociation of PLB from SERCA
-faster reloading of calcium in stores
-increased ATP consumption
-increased chronotropy and inotropy

Question 16

what does cAMP and calcium signalling interaction maximise?

Answer 16

contractile response from cardiac cells

Question 17

how are cAMP FRET sensors used to study cAMP in human bronchial epithelial (HBE) cells and cystic fibrosis?

Answer 17

??

Question 18

what does ezrin (AKAP) bind?

Answer 18

to PJA and targets it to CFTR
-requires NHERF1 as an adaptor

Question 19

what does over expression of NHERF1 cause?

Answer 19

-rescuing of F508del CFTR expression in plasma membrane of CF airway cells by promoting F actin organisation
-through NHERF1-ezrin-actin-complex

Question 20

where is PKA activity highest?

Answer 20

-plasma membrane due to higher cAMP concentration
-local cAMP concentration rather than absolute PKA content determines level of PKA activity in the two compartments

Question 21

what is the role of actin cytoskeleton in cAMP micro domains?

Answer 21

??
suggests actin cytoskeleton and/or NHERF1 could be new targets for CF drug development (stabilisers) esp for the mutations that decrease stability of mutant CFTR in the plasma membrane