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Year 2 Semester 1 > PSC2002 Membrane Transport and Cell Signalling > Flashcards

PSC2002 Membrane Transport and Cell Signalling Flashcards

1
Q

Name some components of the cell membrane

A

-Phospholipid bilayer
-Gprotein coupled receptor
-Channels
-Membrane proteins

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2
Q

Describe Macrotransfer across a cell membrane and some examples.

A

-Large scale movement of molecules
eg -Endocytosis (membrane wraps around stuff and brings it into the cell)
-Exocytosis (vesicles fuses with membrane to release substances)

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3
Q

Describe Microtransfer across a cell membrane and an example.

A

-Smaller scale movement of molecules
eg -Cell membrane transport of small molecules and ions

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4
Q

What is vesicular transport involved in?

A

Endocytosis and Exocytosis through the cell surface membrane

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5
Q

Describe the decreasing plasma membrane permeability of certain molecules

A

-Hydrophobic molecules
-Small uncharged polar molecules
-Large polar molecules
-Ions

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6
Q

Describe the resting membrane potential of all cells

A

-Determined by distribution of ions across the membrane
-Mostly due to Na+ and K+
-Determined by the Nernst equation

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7
Q

Outline the Nernst equation

A

Em = (RT / zF) * ln([ion conc outside] / [ion conc inside])

Em = Membrane potential
RT = Gas constant x Temperature
zF = Number of charges on ion x Faraday’s number

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8
Q

What does the Nernst equation predict?

A

It predicts equilibrium membrane potential based on concentration gradient of that ion across that membrane

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9
Q

Describe the permeability ratio of K+:Na+ in Non excitable cells and nerve/muscle cells.

A

Non-excitable cells 2:1
Nerve/Muscle cells 25:1

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10
Q

Describe how a voltage clamp works

A

-A specific membrane potential (voltage) is set
-The clamp measures how much current (ionic flow) is needed to keep the membrane potential al that set value
-Despite natural changes in ion concentration and permeability

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11
Q

What channel properties can a voltage clamp tell us?

A

-Activation and inactivation kinetics in response to voltage changes
-Reversal potential (MP at which there is no net ionic current through a particular ion channel, indicating the ion’s equilibrium potential)
-Pharmacological effects

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12
Q

What are the main applications of the voltage clamp to study membrane potential?

A

-Ion channel analysis
-Pharmacology studies

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13
Q

What are the main applications of the current clamp to study membrane potential?

A

-Action potential studies
-Excitability studies

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14
Q

Describe how current clamp works

A

-A constant or variable current is injected into the cell
-The resulting changes in MP are measured
-Indicating how the cell responds to the injected current (including action potential firing, threshold potential)

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15
Q

Describe how patch clamping works

A

-Involves using a micropipette to form a high resistance seal with the cell membrane, allowing for measurement of ionic currents
-Can isolate the activity of individual ion channels

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16
Q

Describe the method by which molecules move directly across the lipid bilayer

A

-Passive (simple) diffusion
-Solute/gas passes down concentration gradient
-At finish inward flux = outward flux
-Obeying Fick’s law of diffusion

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17
Q

What methods of moving across the phospholipid bilayer are there using integral membrane proteins

A

-Facilitated diffusion (through pore, channels and carriers)
-Active transport (requiring ATP to transport)
-Secondary active transport

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18
Q

What are the types of secondary active transport?

A

-Co-transport (Symporters) involve movement of a solute coupled to the movement of another down its concentration gradient
-Counter-transport (antiporters) involve coupled movement of two or more solutes in opposite direction

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19
Q

Describe Fick’s law

A

Flux = Permeability x concentration difference

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20
Q

How do you determine permeability?

A

(Diffusion coefficient x Partition coefficient) / Distance

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21
Q

What are the three types of transporters that facilitate diffusion?

A

-Channels (non gated)
-Gated channels
-Uniporters (carriers)

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22
Q

Name some important integral non gated channels

A

-Porins in bacteria, mitochondria and nuclear pore complex
-Aquaporins

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23
Q

What is ENaC?

A

Epithelial sodium channel

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24
Q

Name some important gated channels

A

-ENaC
-K+ channels
-Ca2+ channels
-Almost all ion channels

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25
Q

Give some functional components of gated channels

A

-Gate
-Sensor (detects signal to open gates)
-Selectivity filter (stops other substances)

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26
Q

What modulates the function of gated channels?

A

-Voltage
-Mechanical stimuli
-Ligand binding

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27
Q

Name some important uniporters (carrier channels)

A

-GLUT1 (RBC)
-GLUT2 (Intestines)
-GLUT4 (Skeletal/cardiac muscle)

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28
Q

Describe the cycle by which uniporters regulate movement through a membrane

A

-Carrier open
-Solute enters and binds
-Outer gate closes
-Inner gate opens
-Solute released
-Inner gate closed

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29
Q

What can inhibit transporters?

A

Structural analogues

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30
Q

Describe how the Na+/K+ pump is electrogenic

A

-It drives 3 Na+ ions out for every 2 K+ ions in
-Contributing 10% electrical potential across cell membrane

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31
Q

Name some examples of P-type ATPases

A

-Na+/K+ pumps
-Ca2+ pumps in SR

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32
Q

What can be used to inhibit the Na+/K+ pump

A

-Ouabain
-Cardiac glycosides

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33
Q

Name the types of domains found in the Na+/K+ pump

A

Intra, trans and extra membrane domains

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34
Q

Name the intracellular domains found in the Na+/K+ pump

A

-Actuator domain
-Phosphorylation domain
-Nucleotide domain

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35
Q

Describe the binding sites for Na+ and K+ in the Na+/K+ pump

A

-3 binding sites
-However K+ ions are larger so Na+ fits into domains easier
-Leading to the 3:2 stoichiometry

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36
Q

Describe the mechanism in the Na+/K+ pump

A

-Na+ enters the channel and causes conformational change to move Nucleotide domain across
-Nucleotide domain phosphorylates P domain, releasing ADP and energy, which issued to shut gate on one side and open gate on other side
-Na+ is released, and K+ enters the now vacant binding site in channel, causing conformational change to release phosphate from P domain through the actuator domain
-Regenerated ATP binds N domain, and energy is used to open gate, causing K+ to exit and Actuator to reset

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37
Q

Describe the Gibbs-Donnan effect

A

-Describes how the presence of impermeable ions influence the distribution of other ions across that membrane.
-Permeable ions will distribute to balance concentration gradients and electrostatic forces.

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38
Q

Why is the Na+/K+ pump needed to counteract the Gibbs-Donnan effect

A

Na+/K+ pump forces positive ions out of the cell, increasing the water potential, preventing too much osmotic pressure (ie water entering the cell and lysing)

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39
Q

What do secondary active transporters use to transport substances

A

-Use the kinetic energy provided by electrochemical gradients as
-One solute is transported down a concentration gradient
-With another solute coupled to this transport

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39
Q

Describe symporters

A

Secondary active transporters involved in the transport of two or more molecules in the same direction

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40
Q

Give an example of a symporter

A

Na+/Glucose transporter (SGLT)

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41
Q

Describe antiporters

A

Secondary active transporters involved in the transport of two or more ions in opposite directions

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42
Q

Give examples (and their functions) of antiporters

A

-NHE (Na+/H+) maintains cytosolic pH
-NCX (Na+/Ca2+) maintains low cytosolic Ca2+ conc

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43
Q

What are ABC transporters?

A

ATP Binding Cassette transporters

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44
Q

Describe ATP Binding Cassette transporters

A

-Utilise energy from ATP hydrolysis to transport various molecules across cellular membrane
-Can remove toxins

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45
Q

Give an example of an ATP Binding Cassette transporter

A

CFTR

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46
Q

How is CFTR an irregular ATP Binding Cassette transporter?

A

As ATP regulates the channel

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47
Q

Describe the domains found in ABC transporters

A

-Two Membrane spanning domains
-Two nucleotide binding domains
-One Regulatory domain

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48
Q

What is Osmosis

A

Passive movement of a solvent to a region of high solute concentration across a semi-permeable membrane

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49
Q

What are the major solutes driving osmosis?

A

IONS

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50
Q

What is osmolarity and its units

A

Total concentration of dissolved particles in a litre of solution (osmol/L)

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51
Q

What is osmolality and its units

A

The number of dissolved particles per unit mass (osmol/kg H2O)

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52
Q

What is osmotic pressure?

A

Pressure exerted by flow of water across membrane, determined by solute concentrations

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53
Q

What is tonicity

A

A measure of the effect a solution has on cells placed in it and is driven by osmolarity

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54
Q

Describe a Hypotonic solution

A

-Has an osmolarity less than intracellular
-Water moves into the cell and it expands

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55
Q

Describe a Hypertonic solution

A

-Has an osmolarity greater than intracellular
-Water moves out of the cell and it shrinks

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56
Q

Describe an isotonic solution

A

-Has an osmolarity that equals intracellular
-No net movement and no change in cell size

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57
Q

Describe the equation to find water flow

A

Water flow = Hydraulic water permeability x Pressure

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58
Q

What protein is involved in the transport of water across a membrane?

A

Aquaporins

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59
Q

If a solute reflection coefficient (σ) is 1, how permeable is the membrane?

A

Absolutely impermeable

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60
Q

If a solute reflection coefficient (σ) is 0.5, how permeable is the membrane?

A

Semi permeable

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61
Q

If a solute reflection coefficient (σ) is 0, how permeable is the membrane?

A

No barrier, it is fully permeable

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62
Q

Describe Epithelial tissues

A

-Line external and internal surfaces
-Internal is known as endothelium, external as epithelium

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63
Q

What lines the lumen of the gut?

A

Epithelial cells making up the epithelium

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64
Q

Where is epithelia found?

A

-Line outside of human body (skin)
-Line internal cavities and lumen of bodies

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65
Q

Give some prominent examples of epithelia

A

-GI tract
-Genito-urinary tract
-Respiratory tract

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66
Q

How many human cancers originate from epithelial cells?

A

85%

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67
Q

Give some functions of epithelia

A

-Protection
-Filtration (lining of kidney tubules)
-Exchange (alveoli)
-Absorption (intestine)
-Sensation (taste buds, olfactory epithelium)
-Secretion (lining of glands)

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68
Q

Give some different epithelial tissue types

A

-Simple squamous
-Simple cuboidal
-Simple columnar
-Stratified squamous
-Pseudostratified columnar
-Transitional

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69
Q

Give the function and location of simple squamous epithelial tissue

A

FUNCTION - Absorption, filtration, minimal barrier to diffusion
LOCATION - Capillaries, alveoli, abdominal and pleural cavities

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70
Q

Give the function and location of simple cuboidal epithelial tissue

A

FUNCTION - Secretion, transportation
LOCATION - Glands and ducts, kidney tubules, ovaries

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71
Q

Give the function and location of simple columnar epithelial tissue

A

FUNCTION - Absorption, protection, secretion
LOCATION - Digestive tract

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72
Q

Give the two types of simple columnar epithelial tissue

A

Ciliated and non-ciliated

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73
Q

Give the function and location of stratified squamous epithelial tissue

A

FUNCTION - Protection
LOCATION - Skin, Mouth, Upper throat, Oesophagus

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74
Q

Give the two types of stratified squamous epithelial tissue

A

-Keratinised (makes impermeable and dry)
-Non-keratinised (kept moist by secretions to prevent from drying out)

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75
Q

Give the function and location of pseudostratified columnar epithelial tissue

A

FUNCTION - Absorption and protection
LOCATION - Upper respiratory tract, trachea

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76
Q

Give the function and location of transitional epithelial tissue

A

FUNCTION - Stretchable layer
LOCATION - Bladder

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77
Q

How do epithelia form a functional unit

A

-Epithelial layers are attached to the extracellular matrix of the basal lamina
-Linked using specialised structures

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78
Q

What structures link epithelial cells to each other and the basal lamina?

A

-Tight junctions
-Anchoring junctions
-Channel forming junctions

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79
Q

Describe tight junctions

A

-Membrane proteins that seal adjacent cells together
-Ensuring molecules cannot leak freely between cells
-Also prevent lateral migration of membrane proteins

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80
Q

By preventing lateral migration of membrane proteins, what do tight junctions in epithelial cells generate?

A

Cell polarity, as distinct cell membrane domains are created

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81
Q

What controls the flow of molecules into epithelial cells?

A

The paracellular barrier, which is established by tight junction proteins

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82
Q

Give an example of an epithelium containing a high vs a low number of tight junction proteins

A

High - Urinary bladder
Low - Proximal tubule

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83
Q

What combination of Claudins that make up a tight junction protein make it tight?

A

Claudin-1 and Claudin-3

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84
Q

What combination of Claudins that make up a tight junction protein make it leaky?

A

Claudin-1 and Claudin-2

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85
Q

Describe anchoring junctions found in epithelia

A

-Provide mechanical stability
-Anchor cells to the basal lamina, or to other cells
-Two categories

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86
Q

What are the two categories of anchoring junctions in epithelia?

A

-Actin attachments
-Intermediate filament attachments

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87
Q

What are the two types of actin attachment anchoring junctions in epithelia?

A

-Cell to cell: Adherens junction
-Cell to basal lamina: Focal adhesions

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88
Q

What are the two types of intermediate filament attachment anchoring junctions in epithelia?

A

-Cell to cell: desmosomes
-Cell to basal lamina: hemidesmosomes

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89
Q

Describe the basal lamina

A

Strong and flexible foundation that underlies all epithelia

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90
Q

How thick is the basal lamina?

A

40-120 nm thick

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91
Q

Name the interactions that form the meshwork of the basal lamina

A

-Laminin
-Type IV collagen
-Entactin
-Perlecan

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92
Q

Describe the basement membrane

A

-Combination of the basal lamina and the reticular lamina
-Anchors epithelial cells to connective tissue below
-Acts as a mechanical barrier
-Important in angiogenesis

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93
Q

What is the function of gap junctions in epithelial cells?

A

-Form intracellular channels (approx 1.5nm in diameter)
-Allowing diffusion between cells and enabling cell to cell communication

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94
Q

What are the functions of Ussing chambers?

A

-Measures resistance of ion transport
-Used to determine what ion channels are present (and their concentration and expression)

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95
Q

Describe Ussing studies of frog skin

A

-Skin dissected and mounted as flat sheet between two chambers containing solution of identical composition
-Skins developed a trans epithelial potential difference due to “active” transepithelial Na+ movement

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96
Q

What do polarised epithelial cells generate?

A

Transepithelial voltage due to a total difference in charge across the cell (basolateral - apical)

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97
Q

Describe the mechanism by which epithelial NaCl and water is absorbed

A

-Na+ influxes through ENaC
-Na+ is actively pumped out of the cells across the basolateral membrane via the Na+/K+ ATPase
-Causing paracellular transport of Cl via tight junctions to maintain electroneutrality
-Increasing the NaCl conc on the basolateral side of the epithelium, driving osmotic movement of water

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98
Q

Name some epithelial tissues containing ENaC

A

-Kidney
-Lung
-Colon
-Sweat gland

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99
Q

Simply, what is the role of ENaC in the kidney?

A

Na+ retention, controlling whole body Na+ and water balance, and therefore blood pressure

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100
Q

What is the role of ENaC in the lung?

A

Na+ and water absorption, controlling the amount of airway surface liquid (ASL) and alveolar lining fluid (ALF)

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101
Q

What is the role of ENaC in the colon?

A

Na+ and water reabsorption from the diet

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102
Q

What is the role of ENaC in the sweat gland?

A

Na+ retention, reabsorption of Na+ by sweat ducts, but this is not followed by water, producing a hypotonic sweat secretion

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103
Q

Describe the degenerin ion channel family, and give an example

A

-“Acid-sensing”
-eg ENaC

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104
Q

Describe the structure of ENaC

A

-Functional channel is a heterotrimer of 3 ⍺, β or γ subunits, coded by 3 genes
-Each subunit has 2 transmembrane domains (forming the pore)
-Extracellular loops are the site for regulation by CAPs and SPLUNC1

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105
Q

What may regulate ENaC?

A

CAPs and SPLUNC1

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106
Q

What is the site for ubiquitination in ENaC?

A

PY motif in C term

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107
Q

How does aldosterone affect final urinary salt composition?

A

-Changes ENaC function in the aldosterone sensitive distal nephron
-Binds to cytosolic mineralocorticoid receptor, which goes to nucleus and binds with HRE genes, increasing SGK

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108
Q

What occurs in principal cells of the aldosterone sensitive distal nephron following stimulation by HRE?

A

-Surface ENaC levels increase by 2-5 fold
-Increasing Na+/K+ ATPase density
-increased ATP supply to support increased Na+/K+ ATPase activity
-Increased K+ excretion across apical membrane via ROMK

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109
Q

Where is the Aldosterone sensitive distal nephron located?

A

In the last third of the distal convoluted tubule (DCT2), the connecting tubule (CNT) and the cortical collecting duct (CCD)

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110
Q

How does aldosterone increase the number of ENaC in the apical membrane?

A

-ALDOSTERONE decreases the rate of retrieval by stimulating SGK1, which phosphorylates NEDD4 preventing it binding to the PY motif and inhibiting ENaC ubiquitination
-Preventing internalisation

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111
Q

What may be the result of excess aldosterone?

A

Hypertension, hypokalaemia and or alkalosis

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112
Q

What is Liddle’s syndrome characterised by?

A

-Mutations in cytoplasmic region of β or γ ENaC
-Leading to hypertension, hypokalaemia and or alkalosis

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113
Q

Which genes are mutated in Liddle’s syndrome?

A

SCNN1A, SCNN1B and SCNN1G

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114
Q

How do the mutations caused by Liddle’s syndrome lead to excessive gain of function in ENaC?

A

Mutations lead to changes in cytoplasmic region of ENaC subunits, preventing NEDD4-2 binding, increasing the number of ENaC

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115
Q

Describe how increased ENaC function causes hyperkalemia in Liddle’s syndrome

A

-Enhanced ENaC function depolarises apical membrane potential
-Electrical gradient for K+ excretion through ROMK is increased so get excessive loss of K+ in the urine leading to low blood K_

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116
Q

Describe the effect of Amiloride

A

-Inhibits ENaC (preventing Na+ transport), lowering blood pressure
-Hyperpolarises the apical membrane potential, reducing K+ loss and preventing hypokalaemia

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117
Q

Where is ENaC expressed in the lungs?

A

-In the apical membrane of surface epithelial and alveolar type II cells
-ie conducting and respiratory airways

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118
Q

What is airway surface liquid (ASL) vital for?

A

Innate defence of the lungs via mucociliary clearance

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119
Q

What is alveolar lining fluid (ALF) vital for?

A

Efficient gas transfer

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120
Q

What mediates ENaC in the conducting airways?

A

↑Channel activating proteases (CAPS)
↓Antiproteases
↓SPLUNC1
↓ATP

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121
Q

Describe the different ways in which each NaCl ion is secreted by epithelial cells

A

Na+ moves paracellularly, Cl- moves transcellularly

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122
Q

What are the two types of chloride ion channels?

A

CFTR and Calcium Activated Chloride Channel (CaCC)

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123
Q

What differentiates CFTR from other ABC transporters?

A

It is an ion channel, not an active pump

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124
Q

What domains make up CFTR?

A

5
-MSD1 and MSD2 (forming the pore)
-NBD1 and NBD2 (bind ATP)
-R domain (PKA phosphorylation site)

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125
Q

Describe the molecular mechanism of CFTR gating

A

-PKA phosphorylation of the R domain induces ATP binding and dimerisation of the NBDs
-Conformational change in NBDs transmitted to MSDs, leading to pore opening
-ATP is hydrolysed and pore closes
-Dephosphorylation by protein phosphates closes the channel

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126
Q

What has been used to demonstrate that CFTR requires both PKA phosphorylation and ATP binding to open?

A

Inside out patch

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126
Q

What may modulate Calcium activated Chloride channels (CaCC)?

A

Calmodulin (CaM) and CAM dependent Kinase (CaMK)

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127
Q

Describe where Calcium activated Chloride Channels (CaCC) are found

A

-Present in the apical membrane of most epithelial cells expressing CFTR (apart from intestinal cells)
-Also found in apical membrane of gland secretory acinar cells (WITH NO CFTR)
-In some endocrine, smooth and skeletal muscle, and neurones

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128
Q

What activates Calcium activated Chloride channels (CaCC)?

A

A rise in cytosolic Ca2+

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129
Q

What are TMEM16A and TMEM16B?

A

Calcium activated Chloride Channels

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130
Q

Describe the structure of TMEM16A (CaCC)?

A

-Contains 10 TMDs, with the pore region being TMD 6-9
-Contains 3 intracellular loop
-Functional channel is made up of TMEM16A dimer

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131
Q

Describe how TMEM16A is activated

A

-Calcium ions bind to glutamate residues in one of the two ⍺ helices of intracellular loop 3
-Causing ⍺ helices to move apart, opening the pore, enabling chloride transport

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132
Q

What are the two main mechanisms by which HCO3- is secreted?

A

-Directly through the chloride channel itself
-Indirectly via coupling the chloride channel with an apical chloride/bicarbonate exchanger

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133
Q

What apical anion exchanger is involved in NaHCO3 secretion?

A

SLC26A, a Cl-/HCO3- exchanger

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134
Q

Describe how a net NaHCO3 yield secretion occurs in the epithelia?

A

A chloride channel is coupled to SLC26A

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135
Q

As well as containing SLC26A, what else do many epithelia that secrete HCO3- have?

A

Na+ dependent HCO3- cotransporters (NBCs) on the basolateral membrane, supplying cytosolic HCO3- for the SLC26A exchanger

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136
Q

How does PKA phosphorylation of CFTR influence other channels?

A

-Switches on anion exchanger activity for SLC26A
-By physically interacting via the scaffold protein CAP70

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137
Q

Which epithelial tissues does HCO3- secretion occur in (requiring regulation by CFTR)?

A

-Small intestine
-Biliary tract
-Exocrine pancreas
-Airways
-Female and Male reproductive tracts

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138
Q

Which epithelial tissues does HCO3- secretion occur in (not requiring regulation by CFTR)?

A

The stomach

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139
Q

Describe acinar cells in the exocrine pancreas

A

Secrete a variety of digestive enzymes and a low volume, NaCl rich fluid into the ducts, using TMEM16A channels

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140
Q

Describe duct cells in the exocrine pancreas

A

Transport digestive enzymes to small intestine. Produce a high volume, NaHCO3 rich secretion using both CFTR and SLC26A6

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141
Q

What is fluid secretion in the GI tract driven by?

A

Both NaCl and NaHCO3 secretion with CFTR as the dominant anion channel

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142
Q

What mechanisms carry out fluid absorption in the GI tract?

A

-ENaC mediated fluid absorption in the colon
-Sodium linked absorptive ion transporters (SLC26A, NHE3)
-Nutrient absorptive transporters (SGLT1, GLUT5)

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143
Q

Describe how vibrio cholerae infection causes secretory diarrhoeas

A

Cholera toxin inhibits NaCl and fluid absorption (from villi) and stimulates CFTR mediated Cl-/HCO3- and fluid secretion (from crypts)

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144
Q

Describe how Cholera toxin overstimulates CFTR?

A

CT causes ADP ribosylation of G protein that blocks GTP hydrolysis by the subunit, becoming permanently active (producing lots of cAMP)

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145
Q

What may be given to treat secretory diarrhoeas?

A

-Oral Rehydration Therapy
-Isomolar or hypoosmolar salt solution containing NaCl, NaCitrate, KCl and glucose

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146
Q

What pattern of inheritance describes Cystic fibrosis?

A

Autosomal recessive

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147
Q

What is cystic fibrosis caused by?

A

Loss of function mutations in the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene

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148
Q

How many people in the UK live with CF?

A

Over 11,000

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149
Q

How many people in the UK are carriers for cystic fibrosis?

A

1 in 25

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150
Q

How many mutations in the CFTR gene cause CF?

A

720

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151
Q

What is the most common mutation in the CFTR gene leading to CF?

A

-F508del, loss of phenylalanine at position 508 in NBD1
-50% are homozygous for F508del, and 35% contain a single F508del allele

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152
Q

What are the two types of CFTR mutations?

A

-Minimal function mutations
-Residual function mutations

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153
Q

What are the different classes of CFTR mutations for those with CF?

A

-No protein (class I)
-No traffic (class II)
-No function (class III)
-Less function (class IV)
-Less protein (class V)
-Less stable (class VI)

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154
Q

Give an example of a class I CFTR mutation

A

G542X

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155
Q

Give an example of a class II CFTR mutation

A

F508del

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156
Q

Give an example of a class III CFTR mutation

A

G551D

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157
Q

Give an example of a class IV CFTR mutation

A

R117H

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158
Q

Give an example of a class V CFTR mutation

A

A455E

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159
Q

Give an example of a class VI CFTR mutation

A

rF508del

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160
Q

Which classes of CFTR mutations produce the most severe phenotype?

A

Classes I, II and III
ie Minimal function mutations

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161
Q

Can Cystic fibrosis phenotype severity be predicted from genotyping?

A

-Sometimes, depending on which organ
-Lung disease severity is more variable (influenced by environment, other genetic modifiers)
-Pancreatic insufficiency can correlate well with genotype

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162
Q

How does cystic fibrosis affect the function of the pancreas?

A

-Defective CFTR leads to reduced chloride and bicarbonate secretions, causing sticky mucus that block ducts from acinar cells
-Leading to
-acidic environment which activates digestive enzymes, digesting the ducts
-blocked enzyme flow to the small intestine

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163
Q

What is the role of CFTR in conducting airways?

A

-Maintains proper hydration and pH of the airway surface liquid (ASL), which induces the PCL layer and the mucus layer
-Ensuring efficient mucociliary clearance

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164
Q

Name some cell types in the conducting airway epithelium

A

-Club cell
-Goblet cell
-Basal cell
-Ciliated cell
-Ionocyte
-PNEC
-Tuft cell

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165
Q

How does cystic fibrosis affect mucociliary clearance?

A

-Airway surface liquid is dehydrated and more acidic leading to viscous mucus, mucus accumulation and obstruction, and failure of MCC
-Leading to chronic bacterial colonisation and the establishment of biofilms
-Leading to destruction as chronic immune response in this area

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166
Q

How does cystic fibrosis lead to more acidic airway surface liquid?

A

-Lack of CFTR function reduces HCO3- secretion into the ASL
-H+ secretion is active, producing more acidic ASL

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167
Q

What are the consequences of a more acidic ASL?

A

-Increases fluid absorption due to enhanced ENaC activity (increased CAP and decreased SPLUNC1)
-Increased mucus stasis and viscosity (decreased mucin release)
-Decreased bacterial killing (reduced AMP activity)

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168
Q

What are the common symptoms that characterise cystic fibrosis in the lungs?

A

-Mucus clogging
-Reduced airway surface liquid
-Recurrent lung infections
-Overactive immune response
-Destruction of lung

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169
Q

How is mucus clogging in the lungs in cystic fibrosis treated?

A

Using physiotherapy and mucolytics

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170
Q

How is reduced airway surface liquid in the lungs in cystic fibrosis treated?

A

-Using nebulised hypertonic saline or mannitol
-Drawing water from the body into the airways, improving hydration

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171
Q

How is overactive immune response in the lungs in cystic fibrosis treated?

A

Using NSAIDs

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172
Q

What are the main approaches to the treatment of the basic anion permeability defect in cystic fibrosis?

A

-CFTR modulator therapy
-Genetic therapy
-Alternate channel therapy

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173
Q

In terms of CFTR function, how much function is required to reduce symptoms?

A

<5% is involved in lung disease, meaning giving little CFTR function improves quality of life significantly!

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174
Q

How may we assess cystic fibrosis therapies in patients?

A

-Lung function tests eg FEV1
-Sweat chloride test
-Other relevant parameters such as BMI, quality of life, number of hospitalisations

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175
Q

Give some key structures found in a sweat gland

A

-Secretory coil secretes NaCl rich isotonic from acinar cells (mostly Cl secretion via TMEM16A)
-Absorptive duct absorbs NaCl transcellularly (but no water) producing a hypotonic fluid, using ENaC and CFTR

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176
Q

What regulates ion and fluid transport in healthy sweat glands?

A

Via ACh and Catecholamines

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177
Q

Describe the sweat test for Cystic fibrosis severity?

A

-Sweat glands are stimulated and sweat is collected for 30 minutes
-Sweat chloride greater than 60mmol/Ln indicates Cystic fibrosis
-As NaCl is not reabsorbed in absorptive duct due to faulty CFTR

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178
Q

Describe CFTR modulator therapies

A

-Uses drugs to correct mutant CFTR
-Can be standalone or used in combination with genetic therapies or alternate channel therapy
-Can be personalised

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179
Q

What aspect of CFTR activity can be altered using drug therapies?

A

-Increase the number of CFTR channels at cell surface (class II mutation)
-Enhance CFTR channel gating (class III mutation)
-Increase ion flux (class IV mutation)

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180
Q

What are the CFTR drug modulator types that are currently in clinical use?

A

-Potentiators
-Correctors

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181
Q

What are the CFTR drug modulator types that are currently in development?

A

-Termination suppressors
-Amplifiers
-Stabilisers

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182
Q

Describe what CFTR drug modulator “potentiators” do, and give an example.

A

-Increase the activity of class III gating mutants and some residual function mutants
-Vertex VX770 Kalydeco or Ivacaftor

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183
Q

How do CFTR drug potentiators work?

A

-By increasing the opening rate or duration of openings
-eg VX770 increases G551D CFTR channel activity, and has demonstrated an increase in FEV1 and decrease in sweat chloride tests

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184
Q

Describe what CFTR drug modulator “correctors” do, and give an example.

A

-Promote processing of class II mutants to the plasma membrane, increasing folding efficiency of the channel in the ER
-This increases the number of channels
-VX809 lumacaftor

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185
Q

What have Ussing chambers demonstrated with VX809?

A

VX809 increases F508del CFTR at the apical plasma membrane of human airway cells in vitro, increasing chloride secretion following a cAMP agonist

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186
Q

What defects need to be corrected in F508del CFTR?

A

-Processing defect (low Number)
-Gating defect
-Shorter resident time in plasma membrane (stability)

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187
Q

How do we correct all the defects in F508del CFTR, in order to treat those with CF?

A

-Combination therapy of potentiator (VX770) with corrector (VX809)
-or triple therapy of 2 correctors and 1 potentiator

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188
Q

What is the issue with the current clinical treatments for cystic fibrosis?

A

-They only treat symptoms
-Do not prevent the gradual loss in lung function in most people with cystic fibrosis

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189
Q

Describe what termination suppressor drug modulators for CFTR aim to do?

A

-Work by suppressing nonsense mutations inducing a stop codon
-PTC124 is one currently under development

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190
Q

Describe what amplifier drug modulators for CFTR aim to do?

A

-Work by enhancing stability and transaction of CFTR mRNA
-PTI428 is one currently under development

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191
Q

Describe what stabiliser drug modulators for CFTR aim to do?

A

-Work by increasing the functional stability of CFTR protein in the cell membrane by anchoring it

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192
Q

Define genetic therapies

A

Therapeutic approaches that
-Deliver copies of the healthy gene using gene addition (DNA or mRNA)
-Fix the chromosomal DNA using genome editing (eg CRISPR/Cas9)

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193
Q

What vectors may be used for genetic therapies for cystic fibrosis?

A

Viral or liposome vectors

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194
Q

Describe experimental gene therapies using LIPOSOME vectors for cystic fibrosis

A

-Vesicles with lipid bilayers (coupled with cations) encapsulate plasmid DNA containing functional CFTR gene
-Internalised and gene is inserted into DNA and expressed

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194
Q

Describe experimental gene therapies using VIRAL vectors for cystic fibrosis

A

-Uses adenovirus vector
-Internalised in endosome, and corrected CFTR gene is inserted and expressed in the cell

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195
Q

What are the main issues surrounding gene therapies for cystic fibrosis?

A

-Delivery challenges (patients have thick mucus that hinders delivery of vectors)
-Only transient expression (requires repeated treatments)
-Immune responses lead to reduced efficacy and damage associated with repeated treatments
-Difficult to target correct cells
-May disrupt other genes if integrated into DNA

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196
Q

How may we improve the efficacy of gene therapies?

A

-Using lentivirus, AAV for long term stable correction as the gene is integrated into DNA
-Modify virus to make them less immunogenic
-Use other non viral approaches such as nanoparticles

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197
Q

Describe the steps involved in cell therapy for cystic fibrosis

A

-Isolate cells from skin or other tissue
-Generate induced pluripotent stem cells
-Correct the CFTR mutation to wild type
-Differentiate the cells to basal airway stem cells
-Engraft onto the basal membrane

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198
Q

Describe mRNA mediated therapy for cystic fibrosis

A

-Focuses on delivering synthetic messenger mRNA encoding the functional CFTR to cells
-Bypassing the translation step involved in gene therapies

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199
Q

Describe Antisense Oligonucleotide (ASO)-mediated therapy for cystic fibrosis

A

Focuses on binding to CFTR mRNA, suppressing defective mRNA and improving functionality of the translated CFTR protein

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200
Q

What is the focus of alternate channel therapy for cystic fibrosis?

A

-Focuses on alternative chloride Chanels that are present in CF cells to bypass defective CFTR and restore Cl-/HCO3- and fluid transport
-Aims to inhibit ENaC to help reduce salt and fluid absorption

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201
Q

What is the main target for using alternative chloride channels to treat cystic fibrosis?

A

-TMEM16A
-Need a drug that directly activates the channel without involving Ca2+

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202
Q

How may we decrease ENaC activity to treat cystic fibrosis as part of “alternative channels” therapy?

A

-Use amiloride like drugs
-Inhibit proteases that activate ENaC
-Target ENaC regulatory proteins

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203
Q

What are the two main transporter superfamilies?

A

-ATP binding cassettes (ABC)
-Solute carrier (SLC)

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204
Q

Name some important members of the solute carrier (SLC) transporter superfamily?

A

-OAT (Organic anion transporter)
-OATP (organic anion transporting polypeptide)
-OCT (Organic cation transporter)
-MATE (multidrug and toxin extrusion protein)

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205
Q

Do ATP binding transporters import or export?

A

-They can do either
-But never both

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206
Q

Name some ABC efflux transporters

A

MDR1/P-gp

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207
Q

Name some ABC influx transporter

A

-Maltose uptake transporter
-Methionine uptake transporter
-Typically found in prokaryotes

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208
Q

Name some ABC associated with ion channels

A

-CFTR
-SUR1

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209
Q

Where are ABCs typically expressed?

A

In cells with excretory or barrier functions, eg
-Liver, intestine, kidney
-Blood brain barrier, Blood placenta barrier, Blood testis barrier

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210
Q

What characterises most cells expressing ABCs

A

-Polarised, with ABC transporters being expressed either on the apical or basolateral side
-BUT NOT BOTH

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211
Q

What do ABCs protect cells against?

A

Typically against xenobiotics

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212
Q

How many genes in humans are there for ABC transporters?

A

48

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213
Q

How many ABC subfamilies are there in humans?

A

7, A-G

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214
Q

How many domains are there in ABC transporters?

A

-2 Nucleotide binding domains which bind and hydrolyse ATP
-2 Transmembrane domains which bind and transport substrates

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215
Q

How conserved are ABC NBDs and TMDs?

A

-NBDs are highly conserved
-TMDs are less conserved

216
Q

Name the roles of ABC transporters in the intestine

A

-ABCs interfere with drugs entering enterocytes through brush border membrane
-Pumping compounds such as drugs back into the lumen
-Preventing them reaching the hepatic portal vein

217
Q

Name the roles of ABC transporters in the liver

A

-ABCs transport drugs into hepatocytes from circulation across sinusoidal membrane
-ABCs transport drugs across canalicular membrane to bile or across sinusoidal membrane back to blood (for renal excretion) following metabolism

218
Q

Name the roles of ABC transporters in the blood brain barrier

A

ABC transporters at blood brain barrier prevent many potentially toxic compounds reaching brain

219
Q

Describe what ABC knockout mice studies found?

A

Drug accumulation and toxicity in the brain as they are able to cross the blood brain barrier

220
Q

Describe how a multi-drug resistant tumour is developed

A

-Primary tumour contains mostly normal cells and a few MDR cells
-Following treatment, all normal cells are killed, only MDR cells remain
-Relapse occurs as these MDR cells grow

221
Q

Give examples of mechanisms of multidrug resistance in cancer

A

-Decreased uptake by cell
-Increased metabolism of drug in cell
-Alteration in cell target
-Enhanced drug efflux

222
Q

By what mechanisms may cancer cells enhance drug efflux?

A

By expressing more ABC transporters

223
Q

What is P-glycoprotein also known as?

A

MDR1

224
Q

What gene codes for P-glycoprotein/MDR1?

A

ABCB1 gene on chromosome 7

225
Q

Describe the structure of P glycoprotein

A

TMD1 attached to NBD1, which is attached to TMD2, which is attached to NBD2

226
Q

Describe the proposed model of P glycoprotein/MDR1 mediated transport

A

-Substrate binds to binding site, and ATP binds to NBD
-Conformational change causes drug efflux and release of ADP
-ATP is hydrolysed and conformational reset

227
Q

What is the role of P glycoprotein/MDR1?

A

Removal of xenobiotics from cells

228
Q

Describe methods of P glycoprotein/MDR1 removing xenobiotics from cells

A

-Pumps out xenobiotics from enterocytes following initial absorption
-Transports xenobiotics into bile across canalicular membrane
-Prevents access of many xenobiotics to brain
-Transports xenobiotics into lumen of kidney on brush border membrane

229
Q

Where is P glycoprotein/MDR1 located?

A

Expressed in cells with excretory functions (liver, intestine, kidney) or barrier functions (BBB, Blood placenta barrier, Blood testis barrier)

230
Q

What are the effects of P glycoprotein/MDR1 in tissues?

A

-Limited drug absorption
-Active drug elimination
-Limited drug distribution into tissues

231
Q

What have Schinkel AH et al demonstrated with P glycoprotein/MDR1?

A

-Knockout studies in mice
-Viable, fertile and phenotypically normal
-Suffer toxicity with some compounds due to entry into brain
-Show increased absorption and decreased excretion of a number of drugs

232
Q

Give some substrate types for P glycoprotein/MDR1

A

-Anti cancer drugs
-Tyrosine kinase inhibitors
-Steroids
-Pesticides
-Analgesic
-Antibiotics
-Antihistamines

232
Q

Name some substrates that P glycoprotein/MDR1 and CYP3A4

A

-Cyclosporine
-Tacrolimus
-Paclitaxel
-Doxorubicin
-Midazolam

233
Q

Describe how P53 and P glycoprotein/MDR1 interact in multidrug resistance in cancer?

A

-Wild type p53 represses MDR1 via direct DNA binding
-Wild type p53 mediated down regulation of MDR1 via miR34a and LRPPRC
-Mutant p53 cooperates with ETS1 to up regulate ABCB1 expression
-Increasing MDR1 in cells

234
Q

Describe an alternative method to efflux for multidrug resistance via P glycoprotein/MDR1

A

-Lysosomal accumulation of anticancer drugs
-Plasma membrane containing MDR1 buds inwards to form early endoscopes
-As the endosome matures into a lysosome, it becomes increasing acidified
-Drugs enter the cell and lysosome
-Becomes charged under the acidified conditions and trapped in the lysosome and unable to reach the nucleus

235
Q

Describe BSEP transporters

A

-Coded by ABCB11 gene
-“Sister of P glycoprotein”
-Transports bile salts so named Bile Salt Export Pump
-Expressed only in hepatocytes
-Transports some drugs across canalicular membrane
-Aids in multidrug resistance

236
Q

Describe MDR3 transporters

A

-Coded by ABCB4 gene
-Specific translocate (floppase) for phosphatidylcholine
-Into the lumen from the canalicular membrane in hepatocytes
-Genetic. mutations lead to hepatobiliary diseases, affecting anthracycline, vinca alkalid and taxanes.

237
Q

Describe MRP transporters

A

-Multirdrug resistance associated protein
-Encoded by ABCC1
-Range of functions from protection from xenobiotics to channeling ions
-Facilitate the extrusion of glutathione, glucuronate and sulphate conjugates

238
Q

Where are MRP transporters found in the body?

A

-Barriers (Blood placenta barrier, BBB, Blood testis barrier)
-Excretory and secretory (Liver, Kidney, Lungs Intestine)
-Other tissues (Prostate, Muscle, Blood)

239
Q

Describe the structure of MRP1

A

-Contains 3 transmembrane membrane (TMD0, TMD1, TMD2)
-TMD0 and TMB1 are attached, then to NBD1, to TMD2, then NBD2

240
Q

How doe MRP1 and MDR structures differ?

A

MRP1 contains an extra transmembrane domain, TMD0, making it heavier

241
Q

What molecules do MRP1 prefer?

A

Amphiphillic organic ions, eg LTC4

242
Q

What is LTC4?

A

-Leukotriene C4
-High affinity substrate for MRP1
-Involved in inflammation (important for asthma and allergy)

243
Q

How is LTC4 formed?

A

-By conjugation of GSH to LTA4 via catalysis by leukotriene synthase enzyme
-This enzyme is active in eosinophils, monocytes, neutrophils and macrophages

244
Q

Name some Phase II conjugation reactions in the liver?

A

-Conjugation with glutathione
-Conjugation with amino acids
-Methylation
-Glucuronidation
-Acetylation
-Sulfation

245
Q

What phase II products can MRP1 transport?

A

Glutathione conjugates (also uses GSH as a cotransporter)

246
Q

Name some MRP1 xenobiotic substrates

A

-Alkylating agent
-Antibiotics
-Antifolate
-Anthracycline
-Vinca alkaloids

247
Q

Name some MRP1 endobiotic substrates

A

-GSH conjugates
-Folates
-Sulfate conjugates
-Peptides

248
Q

What have Lorico et al demonstrated with MRP1?

A

-Studied MRP1 knockout mice
-No difference in viability or fertility
-Elevated tissue levels of glutathione (GSH)
-Unchanged tissue levels of glutathione in organs expressing no MRP1
-Increases sensitivity to several chemotherapies

249
Q

What have Johnson et al demonstrated with MDR1 and MRP1?

A

-Knockout mass have hypersensitivity to chemotherapies
-However, when only one is knocked out, the other may compensate

250
Q

What regulates MRP1 expression?

A

MYCN (Blanc et al 2003)

251
Q

What did Ferrandis et al 1994 find with MYCN?

A

MYCN amplification and MDR1 over expression are frequently observed

252
Q

What is MRP2 a transporter of?

A

Glucuronides

253
Q

Where are MRP2 transporters found?

A

-Highly expressed on bile canaliculus on hepatocyte
-Also on apical membranes of kidney and intestine

254
Q

What is the role of MRP2?

A

-Transport glucuronides
-Eliminate drug glucuronides in bile (eg morphine)
-Eliminates bilirubin from body

255
Q

How is bilirubin removed from the body?

A

MRP2 transports bilirubin glucuronide from the liver to the bile

256
Q

What is found in patients with Dubin-Johnson syndrome?

A

-No active MRP2 due to mutations
-Have high levels of bilirubin glucuronide
-May cause jaundice in pregnancy or with some drugs

257
Q

Describe SUR1

A

-Encoded by ABCC8
-No transporter role but acts as ATP sensitive regulator of potassium channel
-Important in control of blood glucose by pancreas

258
Q

Describe how SUR1 functions?

A

-Sulfonylureas bind to receptors causing effect on K+ channel, membrane potential becomes more positive opening voltage gated Ca2+ channels
-Rise in intracellular calcium leads to insulin secretion

259
Q

Name some members of the ABCB family of ABC transporters

A

-MDR1
-MDR3
-BSEP

260
Q

Name some members of the ABCC family of ABC transporters

A

-MRP1
-MRP2
-CFTR
-SUR1

261
Q

Name some members of the ABCG family of ABC transporters

A

BCRP

262
Q

Describe BCRP

A

-Breast Cancer Related Protein
-Encoded by ABCG2 gene
-Also known as MXR
-Also expressed in other tissues and relevant to drug excretion

263
Q

Describe the structure of BCRP/MXR

A

-Single spanning transporter
-Contains one transmembrane domain and one NBD
-Forms homodimers (BCRP) or heterodimers (ABCG5, ABCG8)

264
Q

Where is BCRP located primarily?

A

-Lactating breasts
-Also located in liver, kidney, placenta, testis, colon

265
Q

What is the implication of BCRP’s location on healthcare?

A

-Secretion of xenobiotics into milk
-Meaning restriction on use of certain drugs by nursing mothers

266
Q

What is Imatinib a substrate for?

A

BCRP/ABCG2

267
Q

Describe how Chronic myeloid leukaemia (CML) cells become resistant to Imatinib?

A

-CML stem cells appear to have higher levels of ABCG2
-Decreased levels of regulatory miRNA-212 results in increased ABCG2 levels
-ABCG2 transports imatinib

268
Q

Describe how imatinib can help treat cancer

A

-Inhibits BCR ABL (constitutively active tyrosine kinase driving cell division and inhibiting apoptosis)
-Leads to down regulation of BCRP

269
Q

How are ABCG2/BCRP and gout linked?

A

-GWAS showed a genetic polymorphism resulting in an amino acid change in BCRP is risk factor
-Uric acid (which accumulates in joints) is a substrate for BCRP, which unstable forms of these proteins are unable to excrete this.

269
Q

Describe Tyrosine Kinase inhibitors

A

-Target specific signalling pathways deregulated in cancers
-Targeted treatment for cancers
-Non toxic and more specific
-There are risks of acquired resistance

270
Q

What is the mutation in ABCG2/BCRP that leads to gout?

A

-Q141K
-Causes change in NBD structure

271
Q

How do ABC transporters affect drug development?

A

-Drugs must be tested for ABCs as substrates
-As this may effect the ability of treatments

272
Q

Name some strategies to overcome ABC mediated Multidrug resistance?

A

-Novel delivery systems
-Agents that bypass transporters
-Reduce expression of ABCs eg siRNA, miRNA
-Antibodies
-Natural compounds
-Chemical Inhibitors

273
Q

Describe and name MDR1 inhibitors

A

-Inhibitors of MDR1 to overcome multidrug resistance in cancer
-Verapamil (first gen), Valspodar (second gen), Dofequidar (third gen)

274
Q

What is an added benefit of Verapamil (MDR1 inhibitor)?

A

It sensitises MDR cells to chemotherapy

275
Q

Describe collateral sensitivity in the context of MDR1 inhibitors?

A

-Cancer cells overexpress MDR1/Pglycoprotein, and become hypersensitive to certain compounds
-This stimulates GSH efflux
-This offers a strategy to target drug resistant cancer cells ONLY
-As drugs accumulate in these cells and not others

276
Q

What drugs do 5681014 and BSO sensitise MRP1 expressing cells to?

A

Chemotherapies such as vincristine and doxorubicin

277
Q

Name some inhibitors of ABCG2/BCRP

A

-Verapamil
-Ritonavir
-Elacridar
-Novobiocin

278
Q

What are the issues surrounding targeting ABC transporters as a treatment for cancer?

A

-Physiological roles of ABC transporters
-Ubiquitious expression
-Transporter redundancy
-Dose adjustment and monitering when combining with drugs with narrow therapeutic window

279
Q

What aspects of cancer do ABC transporters lead to?

A

-Inflammation
-Cancer stem like cells
-Anti apoptosis
-Sustained proliferation
-Metastasis and invasion

280
Q

Why is cell signalling important?

A

To transfer information
-From environment to cell
-From cell to cell
To enable homeostasis by reacting accordingly to changes

281
Q

When cell signalling fails, what may occur?

A

Pathology

282
Q

What aspects of physiology may cell signalling affect?

A

-Metabolism
-Nervous system
-Physiology
-Pharmacology
-Cell cycle
-Immunology
-Development

283
Q

What are most signals that a cell responds to?

A

-Most are chemical (eg hormones, neurotransmitters, pheromones)
-Some are physical (eg light, heat, pressure)

284
Q

What are the basic features of cell communication

A

-Synthesis of chemical signal
-Secretion/Release
-Reception (cells decode this interaction)
-Response

285
Q

Give some methods of cell to cell communication

A

-Gap junction
-Paracrine and autocrine signalling
-Hormonal
-Neurohormonal
-Neurotransmitter

286
Q

Give some important cell signalling pathways

A

-Steroid hormone
-Ligand gated ion channels
-Cyclic AMP pathway
-Phosphoinositide pathway
-Tyrosine kinase pathway

287
Q

Give the steps in a cell signal processing pathway and examples of components

A

-Chemical signal (hormone)
-Receptor (ion channel linked)
-Transducer (G protein)
-Amplifier (Adenylyl cyclase)
-Secondary messenger (cAMP)
-Effectors (Protein kinases)
-Response element (enzyme)
-Response (Metabolism)

288
Q

What are the basic principles we find in cell signalling processing

A

-Amplification
-Heterogeneity (each pathway component often has multiple forms)
-Information transfer
-Dynamics (responses are dependent on both temporal and spatial aspects)

289
Q

Describe phosphorylation as a method of covalent modification

A

-The addition of a terminal phosphate group of ATP to hydroxyl group of specific amino acid within a target protein (most commonly serine and threonine)
-By a protein kinase, acting on residues lying within a specific consensus motif
-Changing activity or function of the protein

290
Q

What is the difference in types of phosphorylation kinase enzymes?

A

Type II PKA forms a stable interaction with AKAPs, via the R subunits, so it is not free in the cytosol (type I is). When cAMP binds to the R subunits of type II the catalytic subunits are not released (type I is).

291
Q

How many temporal changes may be different in cell signalling?

A

Differences in frequency or duration

292
Q

What is luminescence?

A

Emission of light by a substance not resulting from heat

293
Q

Give some sources of bioluminescence?

A

-Luciferase (firefly tails)
-Aequorin (Jellyfish)

294
Q

What is phosphorescence?

A

-Light emission continuing for a longer time after the excitation source is removed due to energy being trapped in metastable state

295
Q

What is fluorescence?

A

Emission of light by a substance that has absorbed light

296
Q

What is autofluorescence?

A

-Natural emission of light by biological structures when they absorb light (UV or visible)
-Caused by intrinsic fluorophores (eg NADH, Porphyrins, Elastin)

297
Q

Describe the energy changes in fluorescence?

A

-When light is put in, ground state is energised to excited state
-Excited state then transfers to ground state, releasing energy (light)

298
Q

What is fluorescein?

A

-Synthetic compound that exhibits strong fluorescence
-Used in ophthalmology, immunofluorescence and as a tracer in physiology

299
Q

What is the dichroic filter a component of?

A

Widefield fluorescence microscope

300
Q

What are the three critical filters needed for a fluorescence microscope?

A

-Excitation
-Dichroic
-Emission

301
Q

What is the function of the dichroic filter block?

A

Excitation filter - Selects the appropriate wavelength of light to excite the fluorophlore
Dichroic mirror - Reflects excitation light toward the sample and transmits emitted fluorescence toward the detector
Emission filter - Allows only the fluorescence emission light to reach the detector

302
Q

Describe the light path through a dichroic filter block

A

-Light from the source is passed through the excitation filter, isolating the excitation wavelength
-Filtered light is reflected by the dichroic mirror onto the sample
-Fluorescent light emitted is collected and transmitted back through the dichroic mirror and then passed through the emission filter to the detector

303
Q

What is the benefit of confocal fluorescence microscopy over widefield fluorescence

A

Produces higher resolution and contrast images, eliminating background noise and out of focus light

304
Q

How do you calculate resolution?

A

Lateral resolving power = Wavelength of light / (2 x Numerical aperture of lens)

305
Q

What are the principles of stimulated emission depletion imaging?

A

-Surpasses the diffraction limit of conventional light microscopy
-By combining laser beams and precise optical control to restrict fluorescence emission to a sub diffraction sized region
-Enhancing resolution to as fine as 20-50nm (far below 200nm limit)

306
Q

Give some methods to fluorescence label microscopy samples

A

-Small molecule probes eg
-dye chemistry
-antibodies
-Create fluorescent proteins

307
Q

What are the issues with preparing fluorescent labels in microscopy?

A

-Getting probe to target
-Only labelling the target
-Overcoming any sample autofluorescence
-Phototoxicity
-Photobleaching

308
Q

Give some of the issues with using small molecular probes in fluorescence microscopy

A

-Dye chemistry
-Many cannot be fixed
-Few target individual proteins
-Immunofluorescent techniques
-Difficult to use with live cells
-Only cell surface proteins are visible
-Getting label to target requires permeabilisation

309
Q

What are some of the issues surrounding using fluorescent proteins in microscopy?

A

-Not native proteins
-Strong promoters can enhance signal
-May perturb protein function

310
Q

Give some fluorescence microscopy applications

A

-Live organelle/protein tracking
-Measuring interaction/association via colocalisation
-Identify cells that coexpress certain proteins
-Visualise molecular association (via FRET)

311
Q

What is the function of FRET in fluorescence microscopy

A

-Forster Resonance Energy Transfer
-Established to measure the interaction and location of interaction of two proteins or structures
-Can measure very close interactions (1-10nm)

312
Q

Describe how FRET works?

A

-Donor fluorophore is excited by an external light source
-If the acceptor fluorphore is within the FRET distance of the donor and their emission and excitation spectra overlap, the excited donor transfers its energy to the acceptor without emitting a photon
-The acceptor fluorophore emits light at its characteristic emission wavelength after being excited by the energy transfer

313
Q

Which fluorophores are used in FRET?

A

CFP (cyan fluorescent protein) as the donor and YFP (Yellow fluorescent protein) as the acceptor

314
Q

What can FRET be used to study?

A

cAMP signalling

315
Q

What are neurotransmitters?

A

Chemical messengers, released from one neurone (presynaptic cell) acting at a close site on another (postsynaptic cell) to elicit an effect determined by the specific nature of the receptor

316
Q

Give some key neurotransmitters

A

-Acetylcholine (voluntary movement of the muscles)
-Glutamate (excitatory neurotransmitter)
-Dopamine (Motivation, pleasure)
-Serotonin (emotions, wakefulness and temp reg)
-GABA (inhibitory neurotransmitter)
-ATP (neuronal/glial communication)

317
Q

Describe ionotropic channels

A

-Channel opens when neurotransmitter binds to the receptor
-Allowing ions to flow directly across the membrane
-Causing immediate changes

318
Q

Describe metabotropic channels

A

-Ligand binding activates intracellular signalling pathways
-Usually involving G proteins and second messengers
-May modulate ion channels indirectly

319
Q

What are reversal potentials?

A

-Membrane potentials at which there is no net flow of ions through an ion channel
-Driving forces of the electrochemical gradient are perfectly balanced
-ie Concentration gradient = Electrical gradient

320
Q

What is a quanta?

A

A quantum represents the neurotransmitter content of a single synaptic vesicle, which is released into the synaptic cleft

321
Q

When synaptic vesicles spontaneously release neurotransmitters, what are produced?

A

Miniature end plate potentials (MEPPs)

322
Q

Describe Nicotinic acetylcholine receptors

A

-Found at the neuromuscular junction (muscle cells) and between pre and post synaptic cells in the ANS
-Permeable to Na+, K+ and Ca2+
-Erev of -5mV

323
Q

What do nicotinic acetylcholine receptors consist of?

A

5 subunits - 2⍺, 1β, 1𝛅, 1𝛆

324
Q

What subunit does acetylcholine bind to on nicotinic acetylcholine receptors?

A

⍺ subunit, meaning 2 can bind per receptor

325
Q

Name some antagonists for nicotinic acetylcholine receptors

A

⍺-bungarotoxin (snake venom) and curare compounds (plants)

326
Q

Describe what causes Myasthenia Gravis?

A

-Autoimmune disease
-Body produces autoantibodies against nicotinic ACh receptors
-End plate potential can’t generate muscle stimulation

327
Q

What type of receptors are those for GABAa, GABAc, and Glycine?

A

Ionotropic

328
Q

What type of receptors are those for GABAb?

A

Metabotropic

329
Q

What are GABA and Glycine receptor channels permeable to?

A

Cl- and HCO3-

330
Q

What links GABAa, GABAc and Glycine in structure?

A

All pentameric structures

331
Q

What do the subunit composition of GABA receptors dictate?

A

-Receptor properties (sensitivity, conductance, kinetics of opening and closing)
-Cell surface distribution
-Dynamic regulation

332
Q

Describe 𝛿 subunits of GABA receptors

A

-Sensitive with low desensitisation
-Mediates tonic GABAergic currents

333
Q

Describe how benzodiazepines modulate GABAa receptors?

A

-Mediate sedation
-Bind at interface of ⍺/γ2 subunits
-Eases opening of receptors

334
Q

Describe how barbituates modulate GABA?

A

-Increase the affinity of GABA
-Locks GABA receptors in the open state

335
Q

Describe how alcohol modulate GABA?

A

Enhances GABA action

336
Q

What are the agonists for purinergic receptors?

A

P1 - Adenosine
P2Y- ATP (and other nucleotide triphosphate)
P2X ATP

337
Q

What are the functions of purinergic receptors?

A

-Widespread glia and neuronal expression
-ATP released in synaptic vesicles
-An important means of neuronal to glial, and glia-to-glia communication

338
Q

Describe the role of ionotropic glutamate receptors

A

-Transient opening of ion channels allowing net influx of cations
-Generating an excitatory current

339
Q

Describe the role of metabotropic glutamate receptors

A

-Modulatory role in synaptic transmission
-Roles in learning, memory, and pathology (epilepsy, schizophrenia)

340
Q

Name some classes of ionotropic glutamate receptors

A

-AMPA
-NMDA
-Kainate

341
Q

Name which classes of ionotropic receptors Glutamate and Kainate activate?

A

All! :D
AMPA, NMDA, and Kainate

342
Q

Name which classes of ionotropic receptors Kainate activate?

A

Kainate agonist has no effect on AMPA or NMDA

343
Q

Where are ionotropic glutamate receptors localised?

A

-Usually colocalised at synapses
-Can be both synaptic and extrasynaptic
-Can be both pre synaptic (autoreceptors) and post synaptic

344
Q

What cations do ionotropic glutamate receptors transport?

A

-Ca2+
-Na+
-K+

345
Q

Do ionotropic glutamate receptors work alone or together?

A

They do not work alone, and are part of an enormous and complex protein network

346
Q

Describe the structure of ionotropic glutamate receptors

A

-Comprised of 4 subunits (tetramer)
-Three transmembrane domains (TM1, TM3 and TM4) and a reentrant loop (TM2)

347
Q

What are the two types of modifications that AMPAR subunits can undergo to influence receptor kinetics?

A

-Alternative splicing (flip or flop, long or short tail)
-RNA editing (Q/R, R/G)

348
Q

What are the subunits for AMPA receptors called?

A

GluA1 - GluA4

349
Q

In heteromeric (subunit) AMPA receptor channels, what does the presence of edited GluA2 determine?

A

-The current voltage curve
-Ca2+ permeability

350
Q

What can current flow through AMPA receptors be terminated by?

A

-Deactivation (agonist unbinding leading to closure of channel, requiring removal of transmitter)
-Desensitisation (the channel closes whilst agonist remains bound)

351
Q

What do flip/flop variants of AMPA receptors affect?

A

-Receptor kinetics
-ie the speed at which the receptor desensitises and closes

352
Q

What does editing of the Q/R site on AMPA receptors affect?

A

-Ca2+ permeability
-Acting on GluA2 receptors (swapping glutamine for arginine, which renders GluA2 impermeable)

352
Q

Describe Kainate receptors

A

-Ionotropic glutamate receptors
-Involved in synaptic transmission
-Contributes to plasticity and long term potentiation
-Potent agonist is kainate and glutamate

353
Q

Describe NMDA receptors

A

-Ionotropic glutamate receptors
-Involved in synaptic transmission, plasticity and excitatory signalling
-Binds agonist glutamate and coagonist glycine

354
Q

Describe what activates NMDA receptors

A

-Blocked by Mg2+ “cork”
-Upon depolarisation, this cork “uncorks”
-Allowing free movement of Ca2+ and Na+ through the channel

355
Q

Give an example of evidence that NMDA is involved in memory and learning

A

-Rats are treated with NMDA agonist AP5
-Found to impair spatial memory

356
Q

What is spike timing dependent synaptic plasticity

A

-Synaptic plasticity in which the timing of action potentials determines whether the strength of a synapse is increased (LTP) or decreased (long term depression)
-Caused by NMDA receptors (higher calcium influx through triggers LTP, lower triggers LTD)

357
Q

How does calcium lead to specific cellular responses?

A

-Compartmentalisation of Ca2+ entry
-Localisation of Ca2+ binding proteins

358
Q

What is the phosphoinositide pathway an example of?

A

Second messenger model pathway

359
Q

How is intracellular calcium concentration kept low?

A

Through OFF mechanisms

360
Q

Give some cellular responses to rises in intracellular calcium concentration

A

-Changes in ion permeability
-Secretion
-Contraction
-Metabolism
-Fertilisation
-DNA synthesis
-Development

361
Q

Name some OFF mechanisms for maintaining low intracellular calcium concentrations

A

-Ca ATPase pumps (SERCA and PMCA)
-Na+/Ca2+ exchanger
-Mitochondria and other organelles
-Proteins (buffers and sensors)

362
Q

Name some Calcium ATPase pumps

A

-PMCA (Plasma membrane Calcium ATPase)
-SERCA (SarcoEndoplasmic Reticulum Calcium ATPase)

363
Q

Name a key protein buffer against intracellular calcium

A

Parvalbumin

364
Q

Name a key protein sensor for intracellular calcium

A

Calmodulin

365
Q

Why are high levels of intracellular calcium cytotoxic?

A

Calcium binds with phosphates to create the very insoluble aggregate calcium phosphate (what bones are made of)

366
Q

At what plasma calcium concentrations do OFF mechanisms work at?

A

Varying, however from lowest to highest:
-Endoplasmic reticulum
-PMCA
-Na+/Ca2+ exchanger
-Mitochondria

367
Q

Give the intracellular and extracellular concentrations of calcium

A

Intracellular = ~100nM
Extracellular = 2-3mM

368
Q

Name some second messengers in the phosphoinositide pathway, and what they act on

A

-IP3 acts on IP3 Receptors
-cADPR acts on Ryanodine receptors

369
Q

What is the effect of IP3 binding to its receptors?

A

Opens channels in ER or SR, allowing Ca2+ influx into cytosol

370
Q

What is the effect of Ryanodine receptor binding?

A

Opens channels in ER or SR, allowing Ca2+ influx into cytosol

371
Q

What are the three types of Ca2+ signal?

A

-Elementary events
-Intracellular global Ca2+ wave
-Intercellular global Ca2+ wave

372
Q

Describe the phosphoinositide pathway up to the production of IP3 and DAG

A

1 - Chemical signal induces a confirmational change in a 7 membrane spanning receptor
2 - The III cytoplasmic loop of the receptor activates a G protein (Gq)
3 - Gq stimulates amplifying enzyme Phospholipase C
4 - PLC acts on membrane phospholipid PIP2 to give Diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate (IP3)

373
Q

Describe the phosphoinositide pathway following the production of IP3 and DAG

A

1 - DAG acts within the lipid bilayer to stimulate Protein Kinase C, IP3 diffuses into the cytosol where it acts on IP3 receptors (stimulating Ca2+ release)
2 - Onset of the calcium signal is augmented by Calcium induced calcium release (CICR)
3 - Ca2+ can act through a protein kinase or use specific binding proteins (eg calmodulin) or act directly on ion channels

374
Q

What does Phospholipase C-β act on?

A

Acts on PIP2 to produce diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate (IP3)

375
Q

Describe what the calcium signal takes the form of?

A

A series of Ca2+ spikes, with increasing stimulus leading to increasing frequency

376
Q

Describe Calcium Induced Calcium Release

A

-Ca2+ ion spike feed forward to promote Ca2+ release from sarcoplasmic/endoplasmic reticulum
-Causing a calcium wave through the cell
-What is known as a “regenerative” Ca2+ release

377
Q

Describe the components of intracellular calcium signals?

A

A temporal aspect (the spike) and a spatial aspect (the wave)

378
Q

What is the calcium wave a correlate of?

A

The spatial correlate of the spike

379
Q

What is the calcium spike a correlate of?

A

The temporal correlate of the wave

380
Q

Without calcium induced calcium release, what would occur?

A

-Would rely upon diffusion, which is slow and ineffective due to buffering proteins before sensing proteins are stimulated
-Leading to no wave or spike
-CICR helps to saturate buffers

381
Q

Describe the differing distribution of IP3 receptors and Ryanodine receptors

A

-Hepatocytes contain only IP3Rs
-Cardiac myocytes contain mostly RyRs
-Some cells (eg neurones, secretory cells, eggs) contain both IP3Rs and RyRs

382
Q

Which receptor propagates CICR and calcium waves?

A

Both IP3 receptors and Ryanodine receptors

383
Q

What coagonists allow the release of Calcium from SR or ER

A

Agonist = Calcium
Coagonist = IP3 or cADPR

384
Q

Describe the biphasic regulation of cytosolic Ca2+

A

-Low Ca2+ leads to little Ca2+ release (via CICR) but high Ca2+ concentration inhibits calcium ion release

385
Q

Describe the differing types of inhibition of IP3 receptors and Ryanodine receptors

A

IP3 receptors are truly inhibited at high [Ca2+], but RyRs plateau at high [Ca2+] (ie not complete inhibition)

386
Q

What is the action of cADPR on RyRs?

A

Theorised to sensitise RyRs to CICR

387
Q

What do both IP3 receptors and Ryanodine receptors require for functioning?

A

-Calcium binding
-ATP binding (but not hydrolysis)

388
Q

Why do both IP3 receptors and Ryanodine receptors require ATP binding

A

Thought to help regulate, as sick or diseased cells (with low ATP) can not generate Ca2+ signals as they wouldn’t be able to use OFF mechanisms to remove Ca2+

389
Q

Describe Elementary calcium events

A

-Do not propagate to waves
-Limited to certain areas of the cell
-Have physiological events
-Around 5 microns in diameter
-Around 200ms in duration

390
Q

Describe elementary calcium events acting on IP3 receptors

A

-Calcium Puff
-Acting on about 10 receptors

391
Q

Describe elementary calcium events acting on Ryanodine receptors

A

-Calcium Spark
-Acting on about 10 receptors

392
Q

What are elementary calcium release events caused by?

A

Intermediate amounts of chemical stimulus

393
Q

What are smaller than elementary calcium release events?

A

Fundamental events called blips (IP3) and quarks (RyR)

394
Q

Give an example of how elementary calcium release events having a physiological effect

A

-If calcium sparks (in smooth muscle cells) are close enough to K+ channels (activated by Ca2+)
-Leads to opening and hyperpolarisation, leading to muscle relaxation

395
Q

What are intercellular Ca2+ waves propagated using?

A

-Gap junctions
-IP3 AND Ca2+ needs to move through the junction
-Can propagate between different cell types

396
Q

Describe the sequence in the calcium signalling pathway that leads to the breakdown of glycogen into glucose in liver cells?

A

-Calcium binds to Calmodulin sensing protein
-Ca-calmodulin binds to Kinase II, which phosphorylates phosphorylase kinase
-This phosphorylates phosphorylase
-Which converts glycogen into glucose

397
Q

How does higher frequency of calcium spikes increase Kinase II activation?

A

At higher frequencies, more time is spent in its activated state (not deactivating as it doesn’t have enough time)

398
Q

What can influence the output of the phosphoinositide pathways?

A

-Different isoforms
-eg Type I, II and III IP3 receptors
-eg Type I, II, and III RyRs

399
Q

How may cells differentiate in their phosphoinositide pathways?

A

During development, a process of signalsome expression results in the appearance of cell type-specific signalsomes to create the normal output signals used to control particular cellular functions

400
Q

Give the methods to which signalsomes can be remodelled

A

-Phenotypic remodelling (eg phosphorylation, altering transcription rate of component)
-Genotypic remodelling (eg somatic or gremlin mutations alters activity of component)

401
Q

Describe how phenotypic remodelling of the calcium signalsome can increase force of contraction in the heart

A

cAMP dependent reversible phosphorylation of key Ca2+ signalling components (eg VOC, SERCA) enables heart cells to generate larger Ca2+ signals

402
Q

Describe how phenotypic remodelling of the calcium signalsome can aid liver cell regeneration

A

-Down regulation of key Ca2+ signalling components results in lower frequency Ca2+ spikes of greater duration

403
Q

Describe phenotypic remodelling of the calcium signalsome resulting in Alzheimer’s disease

A

-Extracellular plaque deposits of the β amyloid peptide, disrupting synaptic transmission
-β amyloid oligomers increasing Ca2+ entry via the NMDA receptor
-The amyloid precursor protein intracellular domain (AICD) increasing Ca2+ release from stores
-This abnormal amyloid metabolism results in an up regulation of neuronal Ca2+ signalling to induce an initial decline in memory

404
Q

How does amyloid precursor protein domain (AICD) increase calcium within the cell?

A

-Increases Ca2+ leak through RyR
-Decreases calcium buffering by calbindin

405
Q

Describe the differing calcium levels between in healthy vs Alzheimer’s patients

A

Healthy - Calcium remains at 100nM until spikes with long term potentiation. Raises to 300nm in sleep to erase temporary memories during sleep.

Alzheimer’s - Calcium remains at 300nm continuously, meaning any temporary spikes and memories formed are erased instantaneously.

406
Q

Describe how positive feedback is critical for Alzheimer’s disease progression

A

-Bidirectional relationship between Ca2+ signalling and the amyloidogenic pathway
-While the amyloids stimulate an increase in Ca2+, the latter can stimulate the metabolism of APP
-Producing an element of positive feedback

406
Q

Describe how Calcium signalsome genotypic remodelling can result in Brody myopathy

A

-Skeletal muscle genetic disorder characterised by
stiffness and cramp brought on by prolonged Ca2+
elevation and a slowing of relaxation
-Defect results from mutation in SERCA1 pump
-SR unable to refill with Ca2+; cytosolic Ca2+ remains elevated so relaxation impeded

407
Q

Give some examples of Calcium signalsome remodelling in cancerous cells

A

-Altered SERCA pump activity
-Altered Ca2+ release through IP3 receptors
-Altered resting level of Ca2+

408
Q

Describe the sequence in the cAMP signalling pathway?

A

GPCR->Adenylyl cyclase->cAMP->PKA->Cellular response

409
Q

What are the two major types of Adenylyl cyclase

A

-Membrane bound Adenylyl Cyclase
-Soluble Adenylyl Cyclase

410
Q

Describe Membrane bound adenylyl cyclase

A

-Integral in plasma membrane
-9 Isoforms (AC 1-9)
-Convert ATP to Cyclic AMP
-Regulated by GPCRs, calcium/calmodulin, protein kinases or Forskolin

411
Q

What is Forskolin?

A

A plant-derived diterpene (alkaloid) that directly activates most AC isoforms by binding to their catalytic domains.

412
Q

What GPCRs regulate membrane bound adenylyl cyclase?

A

-Activated by Gαs
-Inhibited by Gαi

413
Q

Name some off mechanisms in the cAMP signalling pathway

A

-Inhibit cAMP production
-Breakdown cAMP
-Remove cAMP from cell

413
Q

Describe soluble Adenylyl cyclase

A

-Found in cytoplasm, unassociated with membrane
-Regulated by bicarbonate ions (activating, and calcium
-Involved in metabolic regulation, sperm motility, intracellular pH sensing

414
Q

Name a membrane bound adenylyl cyclase that is regulated by cytosolic calcium

A

Adenylyl Cyclase 8

415
Q

Describe cAMP production inhibition

A

Some GPCR agonists activate the inhibitory G-protein, Gαi which reduces adenylyl cyclase activity, opposing stimulation by Gαs and thereby lowering cAMP levels

416
Q

Describe the breakdown of cAMP

A

-By phosphodiesterases (PDEs)
-Expression of isoform is tissue specific
-Leading to “shaping” the local cAMP signal’s duration, amplitude, and spatial localisation

417
Q

How many isoforms of phosphodiesterases exist?

A

-11 Isoforms (PDE 1-11)
-8 Breakdown cAMP whilst others breakdown cGMP

418
Q

What inhibits PDEs?

A

-Caffeine (increasing cAMP level)
-Cilastazol (causes vasodilation increasing peripheral vascular flow)
-Milrinone (Increases heart rate and isotropy to fight failing hearts)
-Roflumilast (relaxes airway smooth muscle to reduce airway obstruction)

419
Q

Name what removes cAMP from the cell, and an example.

A

-Range of plasma membrane ABC transporters
-e.g MRP4

420
Q

What are some issues with the “linear” model of cAMP signalling, and what do these issues indicate?

A

-Different agonists increase cAMP levels but produce distinct responses in the same cells
-Some ‘physiological’ agonists produce cAMP dependent responses but do not appear to change global cAMP levels

INDICATING that changes in cAMP must be highly localised to spatially distinct areas inside cells (called microdomains) and changes in cAMP are likely to be agonist specific

421
Q

How is cAMP signalling compartmentalised?

A

-GPCRs localised to different regions of the cell
-Restrict diffusion of cAMP from the plasma membrane to cytosol (by phosphodiesterases)
-Target PKA to distinct sites and substrates in cells (using AKAPs)

422
Q

What are AKAPs?

A

A-Kinase Anchoring Proteins (AKAPs)

423
Q

Describe what AKAPs are, and name key aspects of their structure

A

-Scaffolding proteins, regulating spatial and temporal aspects of cellular signalling pathways
-Contains:
-PKA binding (docking) domain
-Targeting domain
-Multiprotein complex formation regions

423
Q

Describe how AKAPs work?

A

-PKA binding domain anchors the PKA holoenzyme to specific locations within the cell (positioning it near its substrates)
-Targeting domain directs the AKAP to certain cellular compartments eg plasma membrane, nucleus, etc
-AKAPs assemble signalling complexes to form signalsomes

424
Q

Which PKAs do AKAPs bind to?

A

PKA type II

425
Q

Which AKAP is involved in CFTR activation by PKA?

A

Ezrin targets PKA to CFTR, requiring an adapter protein (NHERF1)

426
Q

Describe NHERF1

A

-Helps Ezrin target PKA to CFTR
-Contains:
-PDZ1 binding domain that CFTR binds to
-ERM domain that binds Ezrin

427
Q

Describe the two approaches to measuring cAMP dynamics in living cells

A

-FRET, using a cAMP BD which has two different fluorescent proteins (CFP and YFP) attached that undergo FRET, with FRET decreasing when cAMP rises
-Intensity, cAMP BD + fluorescent protein tag, rise in cAMP increases fluorescent intensity

428
Q

Describe the mechanisms of phenotypic remodelling in the cAMP signalsome

A

-Changes in component expression (Upregulation, Downregulation or alternative splicing)
-Alterations in localisation (of separate components of the signalsome)
-Dynamic assembly and disassembly
-Post translational modifications (eg phosphorylation)
-Receptor desensitisation and cross-talk (can integrate multiple signals from other pathways eg calcium)

428
Q

Describe phenotypic remodelling of the cAMP signalsome in cardiac cells

A

-Activation of β-adrenergic receptor elevated cAMP localised to T-tubules and the SR
-Phosphorylation of key proteins involved in excitation-contraction coupling, including LTCCs, Phospholamban and Ryanodine 2 receptors
-Activation of prostanoid receptors led to phosphorylation of enzymes involved in
metabolism as well as transcriptional factors involved in long-term cellular changes.
-Inhibiting PDEs, abolished receptor-dependent cAMP signalling as well as selective
PKA phosphorylation.

429
Q

Describe how phenotypic remodelling by the cAMP pathway interacts with calcium signalling, helping to coordinate cardiac muscle activity

A

The increased force of contraction induced by β-adrenergic stimulation during exercise, or the fight-or-flight response, leads to PKA-dependent phosphorylation of key Ca2+ signalling components: LTCCs, Phospholamban, and Ryanodine 2 receptors
-Enabling heart cells to generate larger Ca2+ signals (increase inortopy), remove Ca2+ quicker into stores during diastole (lustropy) and reload Ca2+ stores better to increase inotropy

430
Q

How does PKA increase LTCC activity?

A

-PKA phosphorylation increases open state probability of individual LTCCs in the sarcolemma
-PKA also recruits more LTCCs to sarcolemma

431
Q

What is the effect of SERCA phosphorylation during phenotypic remodelling by the cAMP pathway?

A

-Activates it
-Increasing Ca2+ cycling
-Increasing ATP consumption
-Increasing chronotropy and inotropy

432
Q

Name protein kinases activated by cGMP

A

Protein Kinase G

433
Q

Name protein kinases activated by Ca2+

A

-PKC
-Ca2+/Calmodulin dependent PKs

434
Q

Describe the steps in the cyclic GMP signalling pathway

A

-Guanylyl Cyclases are activated by either nitric oxide (synthesised by NOS) or extracellular ligands, converting GTP to cGMP
-cGMP either activates PKG, CNG channels, or regulates PDEs
-cGMP is degraded to GMP by phosphodiesterases (PDEs)

435
Q

What are the two types of guanylyl cyclases in the cGMP signalling pathway?

A

-Soluble form (sGC) activated by nitric oxide
-Plasma membrane bound form (pGC) activated by a smaller number of peptide agonists

436
Q

What breaks down cGMP?

A

cGMP-dependent PDEs

437
Q

Name some ligands that may bind to plasma membrane bound guanylyl cyclases

A

-Atrial natriuretic factor (ANP)
-Brain natriuretic factor (BNP)
-C type natriuretic factor (CNP)
-Guanylin

438
Q

Give some examples of cyclic GMP signalling in health and treatments

A

-Nitric oxide is released from endothelial cell, activating sGC/cGMP in VSM leading to vasodilation and fall in BP. NO generating drugs are often used to treat angina
-Viagra, a type 5 cGMP PDE inhibitor causes rise in cGMP, relaxing smooth muscle in some tissues. Used to treat erectile dysfunction and pulmonary hypertension

439
Q

Give some examples of cyclic GMP signalling in disease

A

-Heat stable enterotoxin from E coli in intestine activates pGC/cGMP, causing PKG to phosphorylate and activate CFTR, leading to a secretory diarrhoea
-LPS (endotoxin) from gram negative bacteria, increases expression in inducible NO synthase, causing excessive NO production. Leads to clinical shock due to severe drop in BP

440
Q

What are the 3 groups of protein kinase C?

A

-Conventional (require both DAG and calcium for activation)
-Novel (Require DAG but are calcium independent)
-Atypical (independent of DAG and calcium)

440
Q

What are the the domains that make up protein kinase C?

A

-Regulatory domains
-Including pseudosubstrate region that mimics the substrate, keeping the enzyme inactive by blocking catalytic domain
-Hinge region
-Catalytic domain (consisting of N lobe and C lobe)

441
Q

Describe activation of PKC

A

-Ca2+ binds to C2 domain, PKC then translocates to the PM, binding to DAG via C1 domain
-Interaction with cofactors displace the pseudosubstrate from C4 domain, activating the kinase
-PKC undergoes autophosphorylation, stabilising its active confirmation

442
Q

What can all isoforms of PKC be activated by?

A

Phorbol esters - plant alkaloids (are tumorigenic)

443
Q

What are the two types of Ca2+ or calmodulin dependent protein kinases?

A

-Multifunctional CaM kinases (eg CaMKII) which are widespread in cells and tissues
-Substrate specific CaM kinases (eg CaMKK) which are specialised in specific cells

444
Q

Give some functions of CAMKII

A

-Regulates activity of NMDA receptors by phosphorylating sites on both NR2A and NR2B (AT ligand gated ion channel lectures)
-Enhances IP3 formation by inhibiting inositol polyphosphate 5-phosphotase
-Central role in frequency decoding of calcium signals, as well as in Long term potentiation and Long term depression
-Phosphorylates PLB to control SERCA2 pump

445
Q

Describe protein phosphatases

A

Remove phosphate groups from phosphorylated proteins (Ser/Thr and Try residues)

446
Q

Name some chemical inhibitors of protein phosphatases

A

-Okadaic acid (blocks PP1 and PP2A)
-Cyclosporin A (very specific for calcineurin (PP2B), used clinically for immunosuppression via effects on T cells

447
Q

Describe Okadaic acid as a chemical inhibitor of protein phosphatases

A

-Toxin produced by algae which accumulate in marine sponges and shellfish
-When ingested cause diarrhetic shellfish poisoning
-Caused by
-Increased paracellular permeability
-More active CFTR promoting fluid loss from GI tract
-INHIBITS PROTEIN PHOSPHATASES

448
Q

Describe how phosphorylase kinase requires both PKA phosphorylation and Ca2+ binding to be fully active in skeletal muscle

A

-The 𝛿 subunit is calmodulin (requires calcium to bind)
-γ is the catalytic subunit
-⍺ and β subunit phosphorylation are required, increasing the calcium sensitivity of phosphorylase kinase

449
Q

What blood glucose concentration is found in healthy vs diseased states (diabetes)?

A

Healthy = ~5mM
Diseased = Can rise to ~60mM

450
Q

What two major hormones are involved in blood glucose homeostasis?

A

Insulin and Glucagon

451
Q

What is the effect of hyperglycaemia on the hormones involved in blood glucose homeostasis?

A

-Stimulates insulin secretion
-Inhibits glucagon secretion

452
Q

What processes does insulin stimulate?

A

-Glucose uptake into muscular cells
-Increased glycogenesis in liver
-Decreased gluconeogenesis in liver

453
Q

What processes does glucagon stimulate?

A

-Glycogenolysis in liver
-Gluconeogenesis in liver

454
Q

Describe pancreatic islets of langerhans

A

-Scattered throughout the pancreas
-Make up 1-2% of pancreatic tissue
-Comprise ⍺, β and 𝛿 cells that secrete hormones

455
Q

What hormones do each cell in the islets of langerhans secrete?

A

⍺ = Glucagon
β = Insulin
𝛿 = Somatostatin

456
Q

Name some regulators that increase insulin secretion?

A

-Glucose (MAJOR)
-Amino acids (MINOR)
-Neural cholinergic input (MINOR)
-Gut hormones eg GLP1, GIP (MINOR)

457
Q

Name some regulators that decrease insulin secretion?

A

-Adrenaline
-Somatostatin

458
Q

Name some regulators that increase glucagon secretion?

A

-Amino acids (MAJOR)
-Stress neural input (MINOR)
-Adrenaline (MINOR)
-Cortisol (MINOR)

459
Q

Name some regulators that decrease glucagon secretion?

A

-Glucose (MAJOR)
-Insulin (MAJOR)
-Gut hormones eg GLP1/GIP (MINOR)

460
Q

Give the sequence of events in insulin production

A

-Preproinsulin is produced, a linear polypeptide consisting of a signal peptide, B-chain, C-peptide and A-chain
-Proinsulin is produced by cleaving signal peptide, consisting of the B-chain, C-peptide, and A-chain. The A and B chain are linked by disulphide bonds
-Mature insulin is produced, consisting of the A and B chains linked by two disulphide bonds

461
Q

Describe the insulin molecule and its storage

A

-Consisting of the A and B chains linked by two disulphide bonds, excluding an intra-chain disulphide bond in the A chain
-In storage granules, insulin forms hexamers stabilised by zinc ions (this is biologically inactive)
-Active in monomer form in bloodstream

462
Q

What is the use of C-peptide from insulin?

A

-Byproduct of insulin synthesis
-Not degraded by the liver
-Means it is valuable as a marker for assessing pancreatic beta cell function and insulin secretion

463
Q

Describe β cellular configuration in islets during a basal blood glucose concentration

A

-ATP K+ channel open, with K+ flux
-Membrane hyperpolarised to -60mV
-Ca2+ channel closed
-Low insulin secretion

464
Q

What acts as the “glucose sensor” in β islet cells?

A

-Glucokinase activity
-ie the amount of ATP in cell

465
Q

Describe β cellular configuration in islets during an increased blood glucose concentration

A

-Raised ATP
-ATP K+ channel closed
-Membrane depolarised to -40mV
-Ca2+ channel open, with high Ca2+
-High insulin secretion

466
Q

What is MODY?

A

Maturity onset diabetes of the young

467
Q

What is PNDM?

A

Permanent Neonatal Diabetes Mellitus

468
Q

What is PHHI?

A

Persistant Hyperinsulinaemic Hypoglycaemia of infancy

469
Q

What mutations cause PNDM?

A

-KCNJ11
-ABCC8
-INS

470
Q

Name some insulin sensitive tissues

A

-Liver
-Muscle
-Adipose

471
Q

Describe what happens in each insulin-sensitive tissue postprandially

A

Liver - Glucose converted to glycogen and triglyceride
Muscle - Glucose to glycogen or metabolised by glycolysis
Adipose - Glucose converted to triglyceride

472
Q

What happens to dietary glucose when the liver glycogen stores are full?

A

-Converted to fat (de novo lipogenesis)
-Increased lipogenesis in adipose tissue
-Utilised for immediate energy needs
-Very low density lipoprotein is secreted (VDLD)

473
Q

Describe what happens in each insulin-sensitive tissue post absorptive (fasted)

A

Liver - Produce glucose (from glycogen) and ketone bodies (from fatty acids)
Muscle - Protein to amino acids (for gluconeogenesis) and metabolise fatty acids for fuel
Adipose - Triglyceride converted to fatty acids and glycerol

474
Q

Describe the action of insulin on metabolic pathways in the liver

A

+Increase glycogen synthesis
+Fatty acid/Triglyceride synthesis
+Protein synthesis
-Decreased glycogen degradation
-Gluconeogenesis

475
Q

Describe the action of insulin on metabolic pathways in muscle tissue

A

+Increased glucose transport
+Increased glucose oxidation
+Increased glucose synthesis
+Increased protein synthesis

476
Q

Describe the action of insulin on metabolic pathways in adipose tissue

A

+Increased glucose transport
+Increased Triacylglycerol synthesis
-Decreased Triacylglycerol breakdown release of fatty acids

477
Q

Describe the cellular action of insulin in muscle tissue

A

-Translocation of GLUT4 from vesicles to PM
-Activation of glycogen synthase
-Activation of pyruvate dehydrogenase

478
Q

Describe the cellular action of insulin in adipose tissue

A

-Increased translocation of GLUT4 from vesicles to PM
-Increased fatty acid synthesis
-Increased A-CoA carboxylase and fatty acid synthase
-Decreased hormone sensitive lipase (HSL)
-Increased TAG synthesis

479
Q

Describe the cellular action of insulin in the liver

A

-Activation of glycogen synthase and A-CoA carboxylase (covalent modification)
-Inactivation of Phosphorylase (covalent modification)
-Induced transcription of Glucokinase, A-CoA carboxylase, and fatty acid synthase
-Repressed transcription of G6Pase, and PEPCK

480
Q

Describe PH protein domains

A

-Pleckstrin Homology domain
-Binds to phosphorylated inositol phospholipid in plasma membrane
-Involved in Insulin signalling pathway

481
Q

Describe PTB protein domains

A

-Binds phosphotyrosine (P-Y) residues
-eg in Insulin receptor

482
Q

Describe SH2 protein domains

A

-Src Homology 2 domain
-Binds phosphotyrosine residues surrounded by unique protein sequences
-Involved in insulin signalling pathway as its recognises phosphorylated tyrosine residues (from the insulin receptor’s intrinsic tyrosine kinase)

483
Q

Describe SH3 protein domains

A

-Src homology 3 domain
-Binds specifically to proline-rich regions (eg SOS)
-Links insulin receptor signalling to the activation of Ras/MAPK pathway

484
Q

Describe the insulin receptor

A

-Receptor Tyrosine Kinase (RTK)
-Heterotetramer, made of ⍺⍺ββ subunits
-Consists of Kinase activation loops, juxtamembrane domains, C terminal domain, Ser/Thr domains

485
Q

What is the function of each insulin receptor subunit

A

⍺ - Insulin binding domain
β - Transmembrane and tyrosine kinase

486
Q

Give the functions of each Insulin receptor domain

A

Kinase activation loop - Activation of tyrosine kinase, phosphorylation creates binding site for SH2
Juxtamembrane domain - Phosphorylation creates binding site for PTB
C terminal domain - Regulates IR kinase activity and kinase-adapter protein interactions
Ser/Thr - Inhibits receptor kinase activty

487
Q

What follows insulin binding the the ⍺ subunit of Insulin receptor?

A

1 - Transphosphorylation of β units
2 - Activation of tyrosine kinase activity
3 - Phosphorylation of IR generates binding sites for proteins with PTB and SH2 domains

488
Q

What is SHC in insulin signalling?

A

-SH2 containing adapter protein
-Links activated insulin receptor to Ras/MAPK pathway

489
Q

Describe the structure of Shc (in insulin signalling)

A

Consists of
-PTB domain
-CH1 domain
-SH2 domain

490
Q

Describe Insulin receptor substrate

A

-Adapter proteins that serve as key adaptor molecules in insulin signalling pathway
-Docking platforms propagating signals from activated insulin receptor
-Functions include metabolic regulation, growth and proliferation, tissue specific roles

491
Q

Name the genes that encode insulin receptor substrate, and key types of insulin receptor substrate

A

-IRS1, IRS2, IRS3 and IRS4
-IRS1 primarily involved in metabolic effects eg glucose uptake and glycogen synthesis
-IRS2 primarily involved in metabolic and growth pathways and is critical for pancreatic β cell function

492
Q

What domains do insulin receptor substrates contain?

A

PH domain and a PTB domain, enabling binding to phosphorylated insulin receptor

493
Q

Describe signalling to the MAP-Kinase pathway via GRB2 in the insulin signalling pathway

A

-Grb2 binds to phosphorylated IRS or Shc through SH2
-This activates and uncovers SH3 domain
-Which binds to SOS, which acts as a GDP/GTP exchange factor
-Ras is activated when bound to GTP
-This activates Raf, which through a cascade activates MAP-Kinase

494
Q

What is the function of MAP-Kinase/ERK in the insulin signalling pathway?

A

Drives growth, differentiation, and proliferation

495
Q

Describe signalling to the PI-3-Kinase pathway in the insulin signalling pathway

A

-Insulin binds to IR⍺ subunit, leading to phosphorylation of IRS on tyrosine
-Leads to recruitment of proteins (eg PI3K) to IRS via SH2 domains causing formation of PIP3
-PIP3 recruits AKT/PKB and PDK1 to plasma membrane
-Dual phosphorylation and activation of AKT/PKB
-Leading to phosphorylation of Protein kinase B/AKT substrates

496
Q

Describe how insulin binding to Insulin receptor ⍺ subunit leads to phosphorylation of Insulin receptor substrate on tyrosines

A

-Insulin binds to subunit
-Activating IR tyrosine kinase
-Autophosphorylation of tyrosines
-Recruitments of IRS to IR
-Phosphorlation of IRS on tyrosines

497
Q

Describe phosphatidylinositol 3-kinase (PI3K)

A

-Adds a phosphate group to the 3 position of the inositol ring in phosphatidylinositol, creating PIP3
-Consists of two subunits, being p110 (catalytic) and p85 (regulatory)

498
Q

What is the functions of PIP3?

A

-Insulin signalling
-Cell proliferation
-Apoptosis
-Cell motility
-Immune activation

499
Q

What mediates the degradation of PIP3?

A

PTEN (phosphatase and tensin homologue)

500
Q

What are mutations in PTEN associated with?

A

Cancer and other diseases as it is a tumour suppressor gene (as well as being involved in insulin signalling)

501
Q

What domains make up AKT/protein kinase B?

A

-N terminal Pleckstrin homology domain (PH)
-Central kinase domain
-C terminal Hydrophobic regulatory domain

502
Q

What activates AKT/Protein kinase B?

A

Dual Phosphorylation (by PIP3) on Thr308 (PKD1) and Ser473 (mTORC2)

503
Q

Give some examples of what AKT/Protein kinase B phosphorylates?

A

-Glycogen synthase kinase 3 (inactivating)
-Preventing inhibition of glycogen storage, lipid synthesis and protein synthesis
-AS160 (involved in GLUT4 translocation)
-FOXO1
-BAD

504
Q

Describe the action of AKT/Protein Kinase B phosphorylation AS160

A

-Inactivates it
-Causing Glut4 translocation to plasma membrane
-Glut4 is the main glucose transporter in muscle and adipose tissue

505
Q

Describe the action of AKT/Protein Kinase B in protein synthesis

A

-mTORC1 is major regulator of protein synthesis, and is regulated by TSC1:TSC2 complex (inhibits Rheb by converting GTP to GDP)
-AKT phosphorylates TSC2, inhibiting it
-Rheb-GTP activates mTORC1
-mTORC1 activates elF4E-BP1 and S6K1 leading to translation initiation and ribosome biogenesis

506
Q

Describe the action of AKT/Protein Kinase B on FOXO1

A

-FOXO1 acts as a transcription factor inducing G6PC and PEPCK (gluconeogenesis enzymes) and represses Glucokinase
-AKT increases degradation of FOXO1

507
Q

Describe what causes type 1 diabetes

A

-Polygenic disorder
-Leading to autoimmune detruction of the insulin producing cells

508
Q

Describe what causes type 2 diabetes

A

-Polygenic disorder
-Defects in insulin action (obesity)
-Defects in glucose induced insulin secretion

509
Q

Give some complications found in diabetes patients

A

-Retinopathy (loss of sight)
-Nephropathy (kidney function)
-Peripheral neuropathy (loss of feeling in feet)
-Autonomic neuropathy (affecting cardiovascular, gut and urinary functions)
-Macrovascular complications
ALL CAUSED BY CHRONIC HYPERGLYCAEMIA

510
Q

Why is type 2 diabetes associated with obesity?

A

-Obesity is associated with insulin resistance and compensatory enlargement of islets
-Genetic background determines the extent by which β cels can compensate
-Type 2 diabetes develops once β cell capacity can no longer compensate for insulin resistance

511
Q

What does increased insulin resistance lead to in those with robust β cells?

A

-Increased insulin secretion (compensation)
-Leading to
-Hyperinsulinaemia
-Euglycaemia (GOOD)

512
Q

What are the two types of therapy for type 2 diabetes?

A

-Lifestyle changes (decreasing calorie intake and increasing exercise)
-Drugs (either monotherapy or combination therapy)

513
Q

What are the main targets of drugs for type 2 diabetes

A

-Carbohydrate digestion
-Renal glucose excretion
-Insulin secretion
-Adipose tissues
-The liver

514
Q

Describe how ⍺-glucosidase inhibitors are used to treat type 2 diabetes, and give an example

A

-Slow down digestion and absorption of carbohydrates
-Reduce rate at which complex carbohydrates are broken down into glucose by glucosidases
-Slow the rise in postprandial blood glucose

EXAMPLE: Miglitol

514
Q

Give some benefits and drawbacks of ⍺-glucosidase inhibitors as treatments for Type 2 diabetes

A

+ Decrease intestinal glucose absorption
+ Decrease glycemic index of food
+ Decrease post prandial blood glucose conc
+ Decrease post prandial triacylglycerides
+ No risk of hypoglycaemia
- Causes abdominal discomfort
- Fermentation of undigested carbohydrate in the colon

515
Q

Describe how SGLT inhibitors are used to treat type 2 diabetes, and give an example

A

-Target SGLTs in the kidneys
-Prevent reabsorption of glucose, causing excretion in urine

EXAMPLE: Phlorizin

516
Q

Give some benefits and drawbacks of SGLT inhibitors as treatments for Type 2 diabetes

A

+ Can cause weight loss due to loss of calories in urine
+ Slow the progression of chronic kidney disease by reducing kidney workload
- Increased urinary volume
- Risk of urinary tract infections (due to more sugary environment)
- Risk of genital fungal infections (due to more sugary environment)

517
Q

Describe how Sulphonylureas are used to treat type 2 diabetes

A

-Bind to SUR1 in K+/ATPase channel
-Acting as an antagonist, closing the channel, depolarising the membrane in islet cells, leading to insulin secretion

518
Q

Give some benefits and drawbacks of sulphonylureas as treatments for type 2 diabetes

A

+ Decrease blood glucose
+ Increase insulin secretion independently of blood glucose
- Increased risk of hypoglycaemia
- Increased weight gain
- Increased risk of cardiovascular events

519
Q

How are GLP1R agonists and dipeptidyl peptidase-4 inhibitors (as treatments for type 2 diabetes) similar?

A

Target incretins

520
Q

What are incretins, and give some examples

A

-Intestinal peptides produced in response to food that stimulate insulin secretion
-GIP (glucose dependent insulinotropic peptide)
-GLP1 (glucagon like peptide 1)

521
Q

Where are GLP1/GIP receptors expressed, and what is their action?

A

-Pancreas (Increase insulin secretion, either decrease or increase glucagon)
-Gut (GLP1 can lead to decreased gastric emptying)
-Kidney (GLP1 can decrease Na excretion)
-Brain (Decrease caloric intake)

521
Q

Give the mechanism of action of GLP1 agonists on the islet β cells

A

-Increase cAMP, leading to increased activity of protein kinase A, increased insulin secretion as K+/ATPase channel is shut, forcing depolarisation
-Increased PI3K, leading to activation of AKT/PKB, increasing gene transcription, decreasing apoptosis, and stimulating cell growth

522
Q

What degrades incretins (and describe them)?

A

-DPP4 (dipeptidyl peptidase 4)
-Exist either as membrane-anchored extracellular enzyme and in soluble form

523
Q

Describe how DPP4 inhibitors are used to treat type 2 diabetes, and give an example

A

-Prevent breakdown of incretins
-Allowing them to inhibit glucagon secretion and increase insulin secretion, lowering blood glucose

EXAMPLE: Sitagliptin

524
Q

Give some benefits and drawbacks of GLP1R agonists and DPP4 inhibitors as treatments for type 2 diabetes

A

+ Moderately decrease blood glucose
+ Increase insulin secretion dependently of blood glucose
+ Decrease glucagon secretion
+ No risk of hypoglycaemia
+ Decrease food intake and body weight
- Potential risks for pancreatitis or pancreatic cancer
- Cause gastric discomfort

525
Q

Describe what occurs to adipose tissue in obesity

A

-Fat cell hypertrophy (increase in cell volume)
-Fat cell hyperplasia (increase in cell number)

526
Q

What occurs when adipocytes attain their maximum capacity of lipid storage?

A

Lipid levels are raised in blood, liver, muscle and islet β cells

527
Q

Describe how PPARγ drugs are used to treat type 2 diabetes, and give an example

A

-Favour adipocyte proliferation and further lipid storage (rather than raising lipid levels in blood and other tissues), as well as increasing lipogenesis and sensitivity to insulin
-Preventing damage by lipids in other organs

EXAMPLE: Thiazolinediones

528
Q

Give some benefits and drawbacks of PPARγ drugs as treatments for type 2 diabetes

A

+Chronically lower blood glucose and insulin
+Increases insulin sensitivity in obesity
~Increases bodyweight and adiposity
-Only pioglitazone still in use as others increased liver damage or chance of heart attacks

529
Q

How does metformin work as a treatment for type 2 diabetes

A

-Inhibition of hepatic gluconeogenesis
-Increased insulin sensitivity in liver and periphery
-Increases peripheral glucose uptake and utilisation
-Increases fatty acid oxidation

530
Q

Describe the cellular mechanisms by which metformin decreases gluconeogenesis and increases fatty acid oxidation

A

-Metformin enters cells via OCT1, and accumulates in energised mitochondria
-This inhibits complex I, decreasing ATP and ADP, increasing AMP, leading to AMPK activation
-AMP kinase inhibits PEPCK and G6Pase, inhibiting gluconeogenesis
-AMP kinase also inhibits A-CoA carboxylase, increasing fatty acid oxidation and decreasing fatty acid synthesis

Year 2 Semester 1 (5 decks)

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