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Flashcards in SESSION 2 Deck (56):
1

Describe the regulatory roles of Calcium

Metabolism:
Bone metabolism
Glycogenolysis
Regulation of many metabolic enzymes, e.g. TCA cycle

Hormonal regulation:
Formation/ degradation of cyclic AMp and GMP
Triggering the release of hormones

Membrane- linked function
Excitation- secretion coupling (e.g. Neurotransmitter release)
Action potential generation
Plasma membrane - Vesicles Fusion

Contractile and Motile systems
Muscle myofibrils
Cilia and flagella
Microtubules and microfilaments

Intracellular signalling functions
Protein kinases protease
Gene expression
Apoptosis

2

What makes calcium such a versatile biological signalling agent?

Divalent structure is very important - strongly attracted to negatively charged or polarised molecules

Relatively polarisable/ squishy - bind to a variety of irregular surfaces

Can adopt a wide range of bonding angles co-ordinating 6-8 negatively charged sites

Proteins- binds to exposed oxygen causing a conformational change- wither physical work or results in further signalling

3

Define the Extracellular calcium concentration

The overall free concentration of free Ca2+ is typically about 1 mM (1 x 10-3M)

4

Define the intracellular calcium concentration

Typically 100nM or 10-7 M

Very small cytoplasmic volumes

5

Define micro domains

Short periods of time where the concentration of calcium ions in a specific region of the cell is even higher for up to a few milliseconds

6

What is the large reservoir source of Ca2+ in the cell?

The smooth endoplasmic reticulum serves as a the principle intracellular Ca2+ store (300uM to 1mM)- rapid release store

Ca2+ can be released rapidly to drive a range of cellular processes: contraction in muscle, synaptic release of neurotransmitter, triggering GI secretions

Mitochondria can also act as an intracellular Ca2+ store- buffering excessive levels of Ca2+
- non rapid release store

7

What are the two main sources of Ca2+?

Extracellular Ca2+ with a concentration of about 1mM

Intracellular SER Ca2+ stores with concentration of 300uM to 1mM

8

What are the advantages of having large calcium gradients?

These large gradients mean that relatively large changes in cytosolic 'sink' concentration can be achieved with relatively small movement of calcium

Also this means that relatively little Ca2+ has to be removed from the cytosol to get back to basal level

9

Describe how entry of calcium from the extracellular source is regulated.

The plasma membrane exhibits highly selective permeability to extracellular Ca2+ via three major channel routes:

1) Voltage gates calcium channels (VOCCs)
Open in response to cell depolarisation.
Depolarisation allows Ca2+ to flow down its concentration gradient very rapidly

2) Ligand gated ion channels (LGICs)
Activated by excitatory neurotransmitters - they bind to the channel and open it so Ca2+ flows intracellular
E.g. NMDA channel- also conducts Na+ and K+

3) Store operated channels (SOC)
Operate slowly
Expressed in both excitable and non- excitable cells
Important when SER stores of Ca2+ are depleted - activated by a special Ca2+ sensing protein in the SER
Important in smooth muscle when prolonged states of stable concentration are required

10

Describe how the efflux of calcium from the intracellular sink to the extracellular source is regulated.

Two very important carriers are responsible:

1) Plasma Membrane Ca2+ ATPase (PMCA)
This Ca2+ specific pump uses 1 ATP molecule to transfer 1 Ca2+ ion out of the cytosol
High affinity for Ca2+- affinity is optimised when it binds to Calmodulin a cytoplasmic Ca2+ sensing protein

2) Na+/Ca2+ exchanger (NCX)
Does not use ATP
Utilises the electrochemical energy gradient provided by large concentration of extracellular Na+
Exchanges 3 Na+ for 1 Ca2+
Subsequently gradient restored by Na+/K+ ATPase
Especially active in excitable tissues

If cell depolarises NCX acts in reverse pumping Na+ out and Ca2+ in

11

Describe how entry of calcium from the intracellular source is regulated.

The SER also has its sets of regulatory tools:

1) GPCRs (Gq type) in the plasma membrane
A ligand binds to the Gq receptor
Triggers production of IP3
IP3 diffuse through the cytoplasm and binds with IP3 receptor

2) IP3 receptors in the SER membrane
IP3 binds to the receptor
IP3 channels opens to allow Ca2+ efflux out of the SER

3) Ryanodine receptors in the SER membrane
Ca2+ act as the ligand
RyR channels vary in three major muscle types: smooth, cardiac and skeletal
Smooth and cardiac are triggered by VOCCs- located in the t- tubules
Depolarisation of the t- tube opens the VOCCs allowing influx of Ca2+
Results in large synchronous outward flux of SR Ca2+ - known as Calcium Induced Calcium Release
Skeletal muscle- the T-tubule VOCCs are physically coupled to the RyR receptor

12

Describe how entry of calcium from the intracellular sink back into the intracellular source is regulated.

Smooth Endoplasmic reticulum Ca2+ ATPase (SERCA)
Enables a rapid reestablishment of the basal Ca2+

13

Describe the role of mitochondrial controlled calcium movement

Known as non- rapidly releasable stores
Low affinity and high capacity carrier system is considered to contribute to regulation of microdomains

14

Describe the control of intracellular calcium by two major protein groups

1) Calcium buffers
The presence of the buffer slows the rate of calcium diffusion into the cytosol
Different calcium buffering proteins can bind up yo 50 calcium ions
These include: parvalbumin, calbindin, calsequestrin and calreticulin (the last 2 bind in the SER)
The buffer proteins can reduce the spread of the calcium throughout the cell and its compartments

2) Calcium Sensors
It is not calcium that directly exerted control usually on proteins but is mediated by calcium binding proteins - regulating other proteins
Including: calmodulin, STIMI and CaM
CaM can bind to up to 4 calcium ions - induce a conformational change - enabling it to interact with other proteins
E.g. Modulation of PMCA Ca2+ ATPase
When CaM binds to Ca2+ it can bind with PMCA to increase calcium sensitivity
Increases sensitivity by a factor of about 10 fold

15

What is the proportionate concentration difference in Ca2+ between extracellular fluid and the cytosol the main compartments?

10 000 fold more concentration in extracellular

16

What would you expect to see happen in an excitable cell such as a neurone or that was heavily depolarised?

NCX would start working in the opposite direction- pumping sodium down its concentration gradient out of the cell and calcium into the cell

17

Contraction in smooth and cardiac muscle is primarily driven by Calcium Induced Calcium Release (CICR). Following membrane depolarisation, outline the main steps in CICR that lead to contraction

Depolarisation of the T-tubules
The VOCCs open- allow influx of calcium
Calcium binds with the RyR- large synchronous outward flux of SR calcium into the sarcoplasm

18

In skeletal muscle following membrane depolarisation, CICR is not considered to be the main driver of Ca2+ release that enables contraction.
Following depolarisation how does release of Ca2+ from the SR in skeletal muscle differ from cardiac and smooth muscle?

Structural modification- the T-tubules are directly physically coupled to the RyR receptor
This coupling means when the VOCC open, the RyR also opens
Massive amounts of calcium are released into the sarcoplasm

19

Briefly outlin how calcium buffers act to regulate cytosolic (Ca2+) following increased levels in a cell

The presence of the buffer slows the rate of calcium diffusion into the cytosol
As they damp down the very rapid entry of calcium throughout the cell
Different calcium buffering proteins can bind up yo 50 calcium ions
The buffer proteins can reduce the spread of the calcium throughout the cell and its compartments

20

Name one calcium buffer present in the cytosol and one in the SER

Cytosol:
parvalbumin and calbindin

SER:
calsequestrin and calreticulin

21

Calcium sensors or 'Trigger proteins' serve as very important mediators of Ca2+ signalling
Calmodulin or CaM is a very important example of a Calcium sensor/ Trigger Protein

How many Ca2+ does CaM bind and what key effect does this binding have on its structure that enable it to act as an intermediary for Ca2+ signalling?

CaM binds to up to 4 calcium
They induce a conformational change
Enables CaM to interact with a very wide range f proteins
Many of the proteins that CaM binds and regulates are unable to bind to Calcium themselves, CaM acts as both a calcium sensor and a signal transducer

22

Explain how CaM modulates the activity of PMCA Ca2+ ATPase to increase Ca2+ efflux

CaM binds to PMCA and increases its sensitivity to Ca2+ by 10 fold
This increases the number of Ca2+ pumped out

23

What is signal transduction?

Signal transduction is the transmission of molecular signals from a cell's exterior to its interior

Signalling molecules can enter the cell to act at nuclear/ intracellular receptors

However the majority of signalling molecules cannot freely cross membrane barriers- expert their actions on cell- surface receptors

24

How do G- proteins work?

Basal resting conditions:
The G- protein exists in its heterotrimeric from with GDP bound to the alpha subunit

Activated receptor:
GDP is released by Ga- subunit and a GTP molecule is now binding in its place
Both a- GTP and By subunits are released and can interact with effectors
The effector interaction is terminated by the intrinsic GTPase activity of the a- subunit- hydrolyses GTP to GDP
Affinity of the Ga- subunit. For GBy- subunit then increases and the GaBy heterotrimer is reformed

25

Describe the structure of the G-protein

G-proteins are heterotrimeric - made up of three distinct subunits: alpha, beta and game

The beta and gamma subunits are so tightly bound they function as a single unit

The alpha subunit has a guanine nucleotide binding site. This site binds GTP and then slowly hydrolyses it to GDP

26

What are the Cellular Targets for activated G- proteins?

Gs
Stimulate adenyl cyclase
Gs carries the signal from the receptor bound by the adrenaline ligand by a B- adrenoreceptor
Then activates the effector enzyme adenyl cyclase
This increases levels of cAMP- activates the enzyme protein kinase A
This phosphorylation a range of proteins
Phosphorylation can either increase or decrease activity

Gi
Adenyl cyclase inhibition
Also has effects on ion channels and signalling pathways involved in growth and differentiation

Gq
Interact with the membrane bound enzyme phospholipids C
Causes hydrolysis of a minor plasma membrane phospholipid - PIP2
This generates 2 secondary messengers (InsP3 and DAG)

27

Describe the Secondary Messenger- Generating Effectors for G- Protein Coupled Receptors

Adenylyl Cyclase
Integral plasma membrane protein
Either activated by Gs- interacting with B-adrenoreceptors
Or inhibited by Gi- interaction with a2- adrenoreceptors

Cyclic AMP interacts with PKA
Phosphorylation a variety of other proteins to increase/ decrease their levels of activity

Dopamine
D1- Gs stimulators
D2- Gi inhibitory

Phospholiase C
Responsible for the hydrolysis of a minor plasma membrane phospholipid- PIP2
Generates two secondary messengers: IP3 and DAG
IP3- activation of RyR on ER to allow Ca2+ ti leave the lumen and enter the cell
DAG interacts with PKC


28

Summarise intracellular second messengers and the protein kinase they regulate

Second messenger Protein kinase
Cyclic AMP. PKA
Cyclic GMP. PKG
Diacyglycerol PKC
Ca2+. CaM - Kinase

29

It is an important role of the receptor G- protein effector signalling system to allow amplification.

Explain how amplification is achieved

Activated receptor can cause GTP/ GDP exchange on more than one G- Protein

An activated Ga- GTP/ GBy can activate multiple effector molecules

Effector molecules act catalytically. Thus activation of adenyl cyclase results in conversion of 100s of molecules of cAMP
The opening of an ion channel by a- GTP allows 100s of ions ot move across the plasma membrane

30

Explain how cAMP contributes to signal amplification

Each activates AC generates many cAMP molecules

CAMP molecules stimulate PKA; each PKA phosphorylation many kinases

Each kinases phosphorylation more targets

31

Explain how deactivation is facilitated by a number of aspects of signalling pathways

Once a receptor has productively interacted with a G protein the binding of the agonist molecule is weakened and agonist- receptor dissociation is more likely to occur

Whilst activated, the receptor is susceptible to a variety of protein kinases which phosphorylation the receptor a prevent it activating further G proteins

The active lifetime of a- GTP may be limited by cellular factors which simulate intrinsic GTPase activity of the Ga- subunit

Enzymes activates in the cell are such that the basal state is favoured. Thus the cell contains high activities of enzymes which metabolise second messengers.

32

Define a ligand

A molecule that interacts with receptors

33

Define agonists

A molecule that binds to the receptor and activate it (leading to intracellular signal transduction events)

Example:
- anti- asthma, B2 adrenoreceptor agonists- SALMETEROL
-anaesthesia- u-opioid receptor agonists- MORPHINE


34

Define antagonists

Molecules that bind to the receptor, but do not activate it (block the effects of agonists)

Example:
- cardiovascular (hypertension) - B- adrenoreceptor antagonists- PROPRANOLOL
- anti-schizophrenic - D2- dopamine receptor antagonists- HALOPERIDOL

35

Define affinity

How well a ligand binds to a receptor

36

Define efficacy

The ability to produce a desired result

37

What do sensory GPCRs respond to?

Light
Odours
Taste

38

Describe the common structure of GPCR

Single polypeptide chain
7 - transmembrane spanning regions
Extracellular N-terminal
Intracellular C- terminal

39

What two regions of GPCRS can be responsible for ligand binding?

Transmembrane domains, e.g. Adrenaline

N-terminal regions

40

How do GPCRs cause a change in cellular activity?

Ligand binds to GPCR
Causing a conformational change
An activated GPCR must interact with a G- protein
This interaction activates the G protein by causing GDP to be exchanged for GTP on the a- subunit
Alpha- beta- gamma subunit dissociates
Subunits interact with effector proteins
A- subunit GTPase hydrolyses GTP back to GDP
Subunits reform

41

Explain how pertussis toxin works

Pertussis toxin works by preventing GDP from being exchanged for GTP
-uncoupling from receptor activation
Modifies all G-proteins within the cell

42

Explain how cholera toxin works

Modifies all the alpha subunits
Unable to hydrolyse GTP
GTP bound to the alpha subunit cannot be hydrolysed to GDP (permanent off stage)
Therefore permanent on stage- 20 hours before degradation

43

Give examples of effectors that are enzymes

Adenyl cyclase
ATP --> cAMP

Phospholipase
PIP2--> IP3 + DAG

Phosphoinositide 3- kinase
PIP2 --> PIP3

Phosphodiester are
CGMP--> 5'GMP

44

Describe how Gs coupled receptors regulate adenylyl cyclase

Ligand binds to Gs protein coupled receptor
Receptor activated
Causing GDP to be released and replaced by GTP
Alpha GTP sub unit stimulates adenyl cyclase (increases activity)
More ATP converted to cAMP
PKA activated by cAMP

45

Which pathways do Gs coupled receptors activate?

B- adrenoreceptors
D1- dopamine receptors
H2- histamine receptors

46

Describe how Gs coupled receptors regulate adenylyl cyclase

Ligand binds to Gs protein coupled receptor
Receptor activated
Causing GDP to be released by the alpha subunit and replaced by GTP
Alpha GTP sub unit stimulates adenyl cyclase
Inhibition of adenyl cyclase means no ATP is converted to cAMP
Therefore PKA isn't activated

47

Which pathways do Gi coupled receptors activate?

A2- adrenoreceptors
D2- dopamine receptors
U- opioid receptors

48

How does PKA function?

- 2 R subunits and 2 C subunits
- R regulate the activity of the C subunit
- C catalytic subunits
-cAMP concentration in the cell increase
-cAMP bind to the regulatory subunits
- releasing the catalytic subunits
- available to phosphorylation substrates within the cell
Catalytic subunits phosphorylate target proteins in the cell

49

Describe how Gq coupled receptors regulate phospholipase C

Ligand binds to Gq coupled receptor
Receptor activated
GDP released by alpha- subunit and replaced by GTP
Alpha subunit acts of the effector protein PL
Interacts with its own substrate PIP2
Clever to produce IP3 and DAG
IP3 diffuses through cytoplasm to the IP3 receptor - releasing calcium from its store
PKC activated by increased levels of DAG and calcium

50

Which pathways to Gq coupled receptors activate?

A1- adrenoreceptors
M1- muscarinic receptors
H1- histamine receptors

51

Use inotrophy of the heart as an example of a signalling pathway

Inotrophy- the force with which the heart contracts

Adrenaline and no adrenaline active B1 - adrenoreceptors
These in turn activate Gs coupled proteins
GDP dissociates and GTP attaches
Adenyl cyclase increased the concentration of cAMP
CAMP is dependent on protein kinase
Protein kinase phosphorylates VOCC- allowing calcium to enter
Each time the membrane depolarises and the channels open the calcium concentration increase
Thus a greater contraction

52

Use smooth muscle contraction as an example of a signalling pathway

Sympathetically released noradrenaline can interact with vascular smooth muscle a1- adrenorceptors to cause vasodilation

Sympathetically released acetylcholine can interact with bronchioles smooth muscle - M3 muscarinic receptors to cause bronchoconstriction

PKC dependent mechanism--> increase in contractility of smooth muscle and the activity of both calcium and PKC activity

53

Use modulation of neurotransmitter release as an example of a signalling pathway

Pain relieving properties of morphine
Stimulate neurotransmitter release
Works at pre- synaptic level
Morphine binds to u- opioid receptor
Activate Gi coupled protein
Beta gamma has an effector role- interacts with VOCC
Affecting the neurotransmitter release
Amount of calcium entering the cell reduces
Reducing the amount of neurotransmitter
G beta gamma inhibits types of VOCC reducing influx of calcium ad neurotransmitter release

54

If GTPas was subject to mutation describe what consequences there may be for subsequent transmission intracellular signalling

A mutation would either result in fast hydrolysis (GAP) or slow hydrolysis

Fast hydrolysis- more rapid hydrolysis- decrease the level of downstream signalling
Turned off quicker

Slow hydrolysis- increases the level of downstream signalling
Turn off slower

GAP is a GTPase activating protein that increases the rate at which GTP is hydrolysed to GDP - terminating the signalling event

55

Why does the body utilise signal amplification and what is required for its operation to be successful?

Advantages of amplification:
- good economic use of resources
Few ligands needed to generate a good response
- easy to regulate
The more ligands, the more problematic it is to remove

The effector molecules must be very specific to the targets, otherwise amplification of unwanted targets

56

What do you think is particularly different about the modulation of VOCCs following binding of morphine at the u-opioid receptor compared to the example of inotropic regulation?

G-beta gamma is the one that binds to the VOCC
Since they are inhibitory there is no second messenger produced