cardio

Question 1

where are calcium release channels of the junctional SR located?

Answer 1

under L type calcium channels of the t-tubule surface membrane

Question 2

where are Ca2+ sparks evoked?

Answer 2

mostly evoked at Z lines where dyad/ ryanadine receptor clusters are located

Question 3

how are Ca2+ sparks evoked?

Answer 3

evoked by a single L-type calcium current

Question 4

describe how calcium leads to cardiac contraction

Answer 4

t tubule deoplarisation opens L-type calcium channels
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small Ca2+ entry via LTCC raises Ca2+ in junctional cleft
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Ca2+ activates clusters of RYR2 in the junctional SR
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RYR2 emphasises junctional SR ca2+ into the cytoplasm
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Ca2+ binds to myofilaments and initiates contraction
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after repolarisation Ca2+ is removed by SERCA, NXC, PMCA
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unbinding from myofilaments and relaxation

Question 5

what is SERCA2?

Answer 5

SERCA2 is a p-type ATPase (similar to sodium-potassium ATPase)

Question 6

what regulates SERCA2a?

Answer 6

phospholamban (endogenous inhibitor)

Question 7

describe phosphorylation of phospholamban

Answer 7

phospholamban is phosphorylated by PKA and/or CamKII to releave the endogenous inhibition of SERCA2

Question 8

why is CICR efficient?

Answer 8

because of the dyadic organisation of LTCC and RYR2

Question 9

describe the action of isoprenaline

Answer 9

isoprenaline is a beta 1 agonist that causes bigger calcium transients and twitches in cardiomyocytes as well as a FASTER muscle twitches that peak earlier and decay faster.

Question 10

what does PKA act on?

Answer 10

(beta adrenoreceptors stimulate PKA) which acts on PLB, RYR and LTCC

Question 11

what are the major phosphotases in the heart regulating cardiac output?

Answer 11

PP1 which dephosphorylates PLB and RYR
PP2A which dephosphorylates LTCC, RYR and troponin I

Question 12

what is phospholamban?

Answer 12

a 52 amino acid protein with 5x possible phosphorylation sites

Question 13

what is PKA?

Answer 13

a tetramer consisting of 2x catalytic and 2x regialtory subunits. the regulatory subunits bins cAMP and AKAPs to tether catalytic subunits close to its substrate proteins

Question 14

what are AKAPs?

Answer 14

a heterogenous family of scaffolding proteins which bind directly to PKA as well as different interaction partners (kinases, phosphatases, adenyly cylases etc) to create local signalling hubs

they are responsible for the compartmentalisation of PKA and other signalling enzymes within particular domains

facilitate spatiotemporal regulation of cell signaling in response to cAMP generation

all have a conserved amphipathic helix which interacts with the R sub-unit dimerisation domain

Question 15

where are AKAPs found?

Answer 15

at different locations within the cells
AKAP18 is found in the membrane of cells.

due to their different cellular loactions AKAPs can compartmentalise downstream signalling responses

Question 16

what processes in the heart do AKAPs regulate?

Answer 16

several different processes including:
-Ca influx
-release from and reuptake of Ca into the SR
-myocyte repolarisation

Question 17

what is the function of ryanadine receptors?

Answer 17

responsible for calcium induced calcium release from SR

Question 18

how do RYRs work?

Answer 18

open in response to free cytosolic or luminal calcium concentrations (luminal enhances channel opening), causing calcium release from LTCC in SR

closes at high calcium concentrations

Question 19

where are RYRs located?

Answer 19

RyRs are located very close to L-type Ca channels:
-region between the LTCC and RyR is called the dyadic cleft (because of how it appears under the electron microscope
-estimated diffusion distance for ‘triggering’ Ca is 15 nm
-within 10 µs of LTCC opening the local Ca concentration will be 10-100 µM

Question 20

how are RYRs regulated?

Answer 20

pH, caffeine, NO, kinases (PKA), other proteins

Question 21

what is the structure of the ryanodine receptor?

Answer 21

-2 Mda, largest known ion channel
-homotetramer (4 identical subunits)
-3 isoforms – RyR2 (cardiac)
-“Mushroom” shape
-6 TM domains with a hydrophobic loop buried within the membrane
-large cytoplasmic domain which has binding sites for regulatory proteins including:
PP1, PP2A, PKA/mKAP, Calmodulin, FKBP12.6

Question 22

describe the mAKAP regulation of RYR

Answer 22

mAKAP brings PKA in to close proximity with RYR allowing for phosphorylation and CICR

it also recruits other signalling molecules (PDE4D3 and PP2A) which aid in activation of and resetting the system

this facilitates a rapid response to cAMP and protects the cell from Ca2+ overload by switching PKA off

Question 23

what is AKAP-18-deltas effect on LTCC?

Answer 23

AKAP18a (also known as AKAP15) targets PKA to the L-type Ca channel
AKAP18a contributes to the cAMP-dependent augmentation of L-type Ca currents
this effect requires the membrane-targeting domain of AKAP18a

Question 24

describe the regulation of SERCA by PLN

Answer 24

SERCA pumps Ca back into the SR at the end of a beat
-Phospholamban (PLN/PLB) inhibits SERCA activity by reducing its affinity for Ca
when PLB is phosphorylated by PKA:
- PLB dissociates from SERCA, and forms a homo-pentamer complex
relieves the inhibition on SERCA
-Ca re-uptake into the SR is accelerated; the heart relaxes quicker
-the amount of Ca stored in the SR increases; more available for release during next E-C cycle

Question 25

how is a co-immunoprecipitation done?

Answer 25

• immobilise antibody against target protein on beads coated with Protein A or Protein G
• Protein A/G are bacterial proteins that bind immunoglobulins with high affinity
• mix beads with cell lysate to capture the target protein and any associated proteins
• wash beads extensively to remove contaminants
• analyse by either SDS-PAGE/Western Blotting or mass spectrometry
• determine quantitative changes in amount of known binding partner pulled down in different conditions
• can be used to identify novel binding partners of the protein of interest (MS

Question 26

describe FRET

Answer 26

biophysical approach used to investigateprotein-protein interactions both in vitroand in cells
* measure energy transfer between donor(CFP) and acceptor (GFP (or YFP))fluorophores that have been fused to 2proteins thought to interact
* if the two proteins:
-don’t interact, following excitation of CFPwith violet light, it emits blue light
- do interact, following excitation of CFP withviolet light, the energy is transferred to GFPwhich emits green light

Question 27

in what 2 ways can genes of interest be introduced to a culture?

Answer 27

transient transfection - used with cell lines - DNA plasmids incorporated int liposomes which fuse with plasma membrane delivering recombinant DNA into the cell

viral infection - used with primary cells - virus enters the cell through interacting with a cell surface protein delivering the gene of interest

Question 28

describe qPCR

Answer 28

used to asses gene expresiion and can measure the relative ampunts of specific mRNA between samples

isolates RNA from cells/ tissues and adds primers to target a specific gene. a dye that only fluoresces when bound to dsDNA id also included in the PCR mix - progress of reaction is measured by fluorescence.