lecture 21 - dynamics of cAMP signalling

Question 1

problem for cAMP is that there are no ‘cAMP specific’ reporter dyes

Answer 1

scientists have devised different approach that combines a cAMP binding protein with two different fluorescent dyes that undergo FRET (measure temporal and spatial levels of cAMP)

Question 2

high FRET when

Answer 2

cAMP is low

Question 3

when you increase cAMP you get less yellow and more

Answer 3

cyan colour

Question 4

IBMX gets rid of

Answer 4

PDEs

Question 5

heart cells - why do 2 different GPCR cAMP agonists cause different functional affects

Answer 5

activate beta adrenergic receptor with noradrenaline —–> increase cAMP and increased contractility
stimulate prostaglandin receptor with PGE1 —-> increase cAMP but no affect on contractility

Question 6

heart cells - why do 2 different GPCR cAMP agonists cause different functional affects

Answer 6

beta-AR gives localised increase in cAMP by the t tubules sarcoplasm reticulum area which is where excitation contraction machinery is, as SR is where calcium comes from and voltage gated calcium channels are. specific phosphorylation of RyRs are SERCA
prostaglandin causes localised release in a different part of the cell so no contraction and tends to lead to different changes. effects enzymes in metabolism
if u block PDEs you get a global increase in cAMP

Question 7

PKA dependent phosphorylation of key Ca signalling components

Answer 7

1. L-type calcium channels (LTCCs)
2. phospholamban, SERCA pump regulator
   3.RyRs
   enables heart cells to generate larger calcium signals
   SERCA pump removes calcium enables faster relaxation

Question 8

LTCC

Answer 8

phosphorylation increases activity, requires type 2 PKA

Question 9

phospholamban

Answer 9

binds to SERCA pump - negative regulator of SERCA
PKA phosphorylation and increase in calcium causes dissociation of phospholamban from SERCA allowing it to work better

Question 10

overexpression of NHERF1 rescued f508 CFTR in the plasma membrane of CF airway cells

Answer 10

NHERF1 scaffold protein

Question 11

cAMP agonist stimulates chloride efflux through CFTR channel

Answer 11

in CF majority of f508 gets destroyed by proteosome as its targeted as its misfolded
over expressing NHERF some f508 got retained and stabilised in membrane

Question 12

FRET sensors shows in normal cells response to cAMP agonist had a localised release where CFTR is

Answer 12

in CF cells the release was less localised and more diffused into the cytosol
NHERF1 also helped to restore the localisation of the signal