lecture 22 Flashcards
how do we measure cAMP dynamics in living cells?
-use genetically encoded florescent tenses to study spatiotemporal cAMP dynamics
-utilise cAMP binding domains from PKA or EPAC attached to fluorescent proteins
what are the 2 approaches to measuring cAMP dynamics in living cells?
- FRET
- intensity
what is the FRET approach?
-uses cAMP binding domain with 2 different flourescent proteins (CFP/YFP) attached that undergo FRET
-FRET decreases when cAMP rises
what is intensity approach?
-cAMP binding domain and flourescent protein
-rise in cAMP increases flourescent intensity
what do 2 different GPCR cAMP agonists cause different functional effects in heart cells?
-beta adrenergic and PGE receptors
-beta AR activated - increases contractility
-PGER activation = no effect on contractility
what happens with beta adrenergic receptors?
-elevation in cAMP localised to T tubules and SR
-causes phosphorylation of key proteins involved in excitation coupling like LTCCs, SERCA regulator and RyR2
what to prostanoid receptors lead to?
-phosphorylation of enzymes involved in metabolism and transcription factors
-involved in long term changes in cell
what is found when PDEs are inhibited?
-selective PKA phosphoryaltion
-less regulation of spatial patterns in cAMP signalling
-less functional response from cardiac cells
what is found about phenotypic remodelling?
-specific to each receptor
-dependent on spatial changes in cAMP
-dependent on localisation of downstream signalling molecules - the signalsome
what effects does phenotypic remodelling of cAMP/PKA pathway have?
-increased force of contraction induced by beta-adrenergic stimulation leads to PKA dependent phosphorylation of Ca2+ signalling components
what signalling components are phosphorylated?
- L type calcium channels
- phospholamban, SERCA pump regulator
- ryanodine type 2 receptors
what does the phosphorylation allow heart cells to do?
-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
how does phenotypic remodelling by PKA phosphorylation increase LTCC activity?
-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
what is needed for an increase in LTCC activity?
AKAP-79
what happens when you prevent AKAP2 cardiac cells?
reduced ability to stimulate calcium channels
how does phenotypic remodelling by cAMP/PKA pathway affect the SERCA pump?
-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
what does cAMP and calcium signalling interaction maximise?
contractile response from cardiac cells
how are cAMP FRET sensors used to study cAMP in human bronchial epithelial (HBE) cells and cystic fibrosis?
??
what does ezrin (AKAP) bind?
to PJA and targets it to CFTR
-requires NHERF1 as an adaptor
what does over expression of NHERF1 cause?
-rescuing of F508del CFTR expression in plasma membrane of CF airway cells by promoting F actin organisation
-through NHERF1-ezrin-actin-complex
where is PKA activity highest?
-plasma membrane due to higher cAMP concentration
-local cAMP concentration rather than absolute PKA content determines level of PKA activity in the two compartments
what is the role of actin cytoskeleton in cAMP micro domains?
??
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
