L22 Dynamics of cAMP signalling

cAMP levels do not uniformly increase throughout a cell in response to a cAMP agonist. Instead…

changes in cAMP occur in microdomains and utilise signalsomes for efficient signalling

in the ‘calcium’ field, spatial and temporal changes in intracellular calcium are measured using…

Ca2+ sensitive fluorescent indicator dyes, which can easily be loaded into living cells

what can we use genetically-encoded fluorescent sensors to study

spatiotemporal cAMP dynamics

to measure cAMP dynamics in living cells we can utilise cAMP ‘binding domains’ from PKA or EPAC attached to…

fluorescent proteins

we can measure cAMP dynamics using sensors expressed in living cells using…

plasmids

summarise FRET to measure cAMP dynamics in living cells

• uses cAMP binding domain which has 2 fluorescent proteins (CFP/YFP) attached that undergo FRET

• FRET decreases when cAMP rises

summarise intensity to measure cAMP dynamics in living cells

cAMP binding domain + fluorescent protein tag

• rise in cAMP increases fluorescent intensity

in heart cells, 2 different GPCR cAMP agonists cause…

different functional effects

activation of the beta-adrenergic receptor elevated…

cAMP localised to T-tubules and the SR

phosphorylation of key proteins involved in excitation-contraction coupling included…

• dihydropyridine receptors (LTCCs- L-type calcium channels)

• phospholamban (regulator of SERCA)

• ryanodine 2 receptors

what does phospholamban negatively regulate

SERCA

activation of prostanoid receptors led to…

• phosphorylation of enzymes involved in metabolism

• transcription factors involved in long-term cellular changes

Inhibiting PDEs, abolished receptor-dependent cAMP signalling as well as…

selective PKA phosphorylation

what does Inhibiting PDEs, abolished receptor-dependent cAMP signalling as well as selective PKA phosphorylation illustrate

a key role of PDEs in regulating the spatial patterns of cAMP signals and functional responses from cardiac cells

phenotypic remodelling is specific to each receptor and dependent on…

• spacial changes in cAMP

• localisation of downstream signalling molecules (cAMP signalsome)

what does phenotypic remodelling by the cAMP/PKA pathway interact with

calcium signalling

what does phenotypic remodelling by the cAMP/PKA pathway help coordinate

cardiac muscle activity

the increased force of contraction induced by beta-adrenergic stimulation during exercise (or the fight or flight response) leads to PKA dependent phosphorylation of what key Ca2+ signalling components…

• L-type calcium channels

• Phospholamban

• Ryanodine type 2 receptors

what does PKA-dependent phosphorylation of key Ca2+ signalling components do

• generate larger Ca2+ signals

• remove Ca2+ quicker into stores during diastole

• reload Ca2+ stores better

what does generating larger Ca2+ signals do

• increase triggered calcium via LTCCs

• increased Ca2+ store release via RyR2

  • larger CICR so increased inotropy

what does removing Ca2+ quicker into stores enable

faster relaxation (lustropy)

what does reloading Ca2+ stores better help to increase

inotropy

PKA phosphorylation increases LTCC by…

increasing the open probability (Po) of individual LTCCs

the increase in LTCC activity by cAMP/PKA phosphorylation requires…

an AKAP

other work showed that AKAP-79 was the key AKAP regulating…

phosphorylation of LTCC by PKA

beta-adrenergic receptor stimulation of isolated mouse cardiomyocytes also leads to…

increased formation of ‘super clusters’ of LTCCs

LTCCs were visualised on the surface of the myocytes by attaching…

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

superclusters of LTCCs form by interaction of their C-terminal domains, but only after…

PKA-dependent phosphorylation of the LTCC

phenotypic remodelling by the cAMP/PKA pathway affects the…

SERCA pump

PLB =

phospholamban (negative regulator of SERCA)

PKA phosphorylation, and increases in Ca2+ cause

dissociation of PLB from SERCA

beta-adrenergic stimulation leads to enhanced muscle contraction because…

cAMP/PKA signalling is restricted to sites and substrates directly involved in excitation-contraction coupling

cAMP and calcium signalling interact to maximise…

contractile response from cardiac cells

Spatial cAMP signalling is disrupted in

CF airway cells