SESSION 2

Question 1

Describe the regulatory roles of Calcium

Answer 1

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

Question 2

What makes calcium such a versatile biological signalling agent?

Answer 2

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

Question 3

Define the Extracellular calcium concentration

Answer 3

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

Question 4

Define the intracellular calcium concentration

Answer 4

Typically 100nM or 10-7 M

Very small cytoplasmic volumes

Question 5

Define micro domains

Answer 5

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

Question 6

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

Answer 6

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

Question 7

What are the two main sources of Ca2+?

Answer 7

Extracellular Ca2+ with a concentration of about 1mM

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

Question 8

What are the advantages of having large calcium gradients?

Answer 8

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

Question 9

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

Answer 9

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

Question 10

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

Answer 10

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

Question 11

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

Answer 11

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

Question 12

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

Answer 12

Smooth Endoplasmic reticulum Ca2+ ATPase (SERCA)

Enables a rapid reestablishment of the basal Ca2+

Question 13

Describe the role of mitochondrial controlled calcium movement

Answer 13

Known as non- rapidly releasable stores

Low affinity and high capacity carrier system is considered to contribute to regulation of microdomains

Question 14

Describe the control of intracellular calcium by two major protein groups

Answer 14

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

Question 15

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

Answer 15

10 000 fold more concentration in extracellular

Question 16

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

Answer 16

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

Question 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

Answer 17

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

Question 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?

Answer 18

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

Question 19

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

Answer 19

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

Question 20

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

Answer 20

Cytosol:
parvalbumin and calbindin

SER:
calsequestrin and calreticulin

Question 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?

Answer 21

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

Question 22

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

Answer 22

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

Question 23

What is signal transduction?

Answer 23

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

Question 24

How do G- proteins work?

Answer 24

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

Question 25

Describe the structure of the G-protein

Answer 25

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

Question 26

What are the Cellular Targets for activated G- proteins?

Answer 26

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)

Question 27

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

Answer 27

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

Question 28

Summarise intracellular second messengers and the protein kinase they regulate

Answer 28

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

Question 29

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

Answer 29

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

Question 30

Explain how cAMP contributes to signal amplification

Answer 30

Each activates AC generates many cAMP molecules CAMP molecules stimulate PKA; each PKA phosphorylation many kinases Each kinases phosphorylation more targets

Question 31

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

Answer 31

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.

Question 32

Define a ligand

Answer 32

A molecule that interacts with receptors

Question 33

Define agonists

Answer 33

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

Question 34

Define antagonists

Answer 34

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

Question 35

Define affinity

Answer 35

How well a ligand binds to a receptor

Question 36

Define efficacy

Answer 36

The ability to produce a desired result

Question 37

What do sensory GPCRs respond to?

Answer 37

Light Odours Taste

Question 38

Describe the common structure of GPCR

Answer 38

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

Question 39

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

Answer 39

Transmembrane domains, e.g. Adrenaline N-terminal regions

Question 40

How do GPCRs cause a change in cellular activity?

Answer 40

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

Question 41

Explain how pertussis toxin works

Answer 41

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

Question 42

Explain how cholera toxin works

Answer 42

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

Question 43

Give examples of effectors that are enzymes

Answer 43

Adenyl cyclase ATP --> cAMP Phospholipase PIP2--> IP3 + DAG Phosphoinositide 3- kinase PIP2 --> PIP3 Phosphodiester are CGMP--> 5'GMP

Question 44

Describe how Gs coupled receptors regulate adenylyl cyclase

Answer 44

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

Question 45

Which pathways do Gs coupled receptors activate?

Answer 45

B- adrenoreceptors D1- dopamine receptors H2- histamine receptors

Question 46

Describe how Gs coupled receptors regulate adenylyl cyclase

Answer 46

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

Question 47

Which pathways do Gi coupled receptors activate?

Answer 47

A2- adrenoreceptors D2- dopamine receptors U- opioid receptors

Question 48

How does PKA function?

Answer 48

- 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

Question 49

Describe how Gq coupled receptors regulate phospholipase C

Answer 49

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

Question 50

Which pathways to Gq coupled receptors activate?

Answer 50

A1- adrenoreceptors M1- muscarinic receptors H1- histamine receptors

Question 51

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

Answer 51

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

Question 52

Use smooth muscle contraction as an example of a signalling pathway

Answer 52

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

Question 53

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

Answer 53

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

Question 54

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

Answer 54

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

Question 55

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

Answer 55

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

Question 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?

Answer 56

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