L1-12: Membrane transport Flashcards
What are the different mechanisms of movement across membranes?
Passive diffusion (through membrane)
Facilitated diffusion (Ion channels and uniporters)
Secondary active transport (Co and counter transporters)
Primary active transport (ATP)
What does CFTR stand for?
Cystic Fibrosis Transmembrane conductance Regulator
What diseases do defects in CFTR lead to?
Cystic fibrosis and forms of secretory diarrhoea
What are examples of macrotransfer?
Exo and endocytosis
What are the most permeable molecules that will pass through the plasma membrane?
Hydrophobic molecules (O2, N2, CO2 and steroid hormones)
How is the equilibrium potential determined?
Using the Nernst equation
What molecules is resting membrane potential determined by?
Sodium and potassium ions
What can artificial semi-permeable membranes be used for?
Specifically permeable to one or more ions
Measure charge across the membrane
What does the movement of ions depend on?
The electrochemical gradient
What constants does the Nernst equation use?
Gas constant and Faraday constant
What does the Nernst equation measure?
It predicts the equilibrium potential based on concentration of that ion across the membrane
How can membrane potential be measured when multiple ions are involved?
Using Goldman-Hodgkin-Katz (GHK)
What is the ratio of sodium and potassium in non-excitable cells?
Sodium: Potassium
1:2
Nerve/muscle cells 1:25
How can ion channels properties be measured?
Using voltage clamp
How is a voltage clamp used?
Holding voltage is set
Voltage controlled by electronic feedback circuit
Channels open/close as normal (apparatus compensates for changes)
Voltage then stepped and current required to hold voltage is measured (current=total ionic current flowing across membrane)
Why is voltage clamp useful?
Allows a detailed measurement and analysis of electrical activity across a tissue, cell or artificial membrane mediated by specialised ion chancels and electrogenic carriers
What is a current clamp?
Controls amplitude of injected current via microelectrode and allows voltage to vary
How does a current clamp work?
Current clamp circuit controls amplitude using microelectrode
Amplifier records voltage generated by the cell
Why are current clamps used?
To study how a cell responds when electric current enters a cell
Who was the voltage clamp developed by?
Cole and Marmont 1930s-1940s (developed by Alan Hodgkin and Andrew Huxley 1950s)
What is the patch clamp?
A recording pipette that physically isolates a patch of the membrane
Allows the measurement of current flow through single ion channel
What is the basis of patch clamp?
Formation of a high resistance seal between membrane and micropipette
Giga-seal means current flowing through ion channels in patch can be recorded with minimal noise (could be swamped by background electrical noise)
What are the different types of secondary active transport?
Co-transport - movement of solute coupled to movement of another down its concentration gradient
Counter-transport - coupled movement of two or more solutes in opposite directions
How does driving force allow substances to pass through the membrane?
Using electrochemical gradient (sum of chemical potential energy differences and charge differences)
What are the different equations to calculate the flux and permeability of the membrane?
J = PxΔC
Flux = permeability x concentration difference
P = Dκ/x
Permeability = diffusion coefficient x partition coefficient / distance
What are the 3 types of transporters that facilitate diffusion?
Channels
Gated channels
Uniporters
What are examples of the different integral membrane proteins that allow direct access to the cell?
Porins in bacteria, mitochondria and nuclear pore complex
Aquaporins
What are examples of different gated channels?
ENaC - epithelial sodium channel
K+ channels
Ca2+ channels
Almost all ion channels
What are functional components of gated channels?
Gate
Sensor
Selectivity filter
How are gated channels modulated?
By voltage, mechanical stimuli and ligand binding
What is the mechanism of carrier-mediated facilitated diffusion?
Carrier open
Solute enters and binds
Outer gate closes
Inner gate opens
Solute released
Inner gate closes
How can carrier mediated transport saturation be detected?
Flux much greater that predicted using passive diffusion
Carrier mediated diffusion exhibits saturation kinetics
Transporters inhibited by structural analogues
What are P-type ATPases?
They are pumps that are able to phosphorylase themselves during pumping cycles
What are examples of P-type ATPases?
Na+-K+ pumps
Ca2+ pumps in SR (skeletal and cardiac)
What are the properties of the Na+-K+ pump?
1/3 of cells energy to power
Electrogenic: 3 Na+ out 2 K+ in
10% electrical potential across cell membrane
What is the Na+-K+ pump inhibited by?
By Ouabain (strophantus) and cardiac glycosides from digitalis spp
What is the structure of the Na+-K+ pump?
10 transmembrane domains
Actuator domain
Phosphorylation domain
Nucleotide domains
A,P and N interact
I, II and III cation binding sites
β subunit (can be blocked using ouabain)
Why are more Na+ than K+ pumped?
K+ ions are larger
Na+ fits into domains easier
What is the Na+/K+ pump mechanism?
Na+ enters channel
Conformational change moves N domain across
N domain phosphorylates P domain, releases ADP and energy
Energy used to shut gate on one side and open on other
Na+ released
K+ enters and fills pockets
Conformational change releases phosphate from P through the actuator domain
Regenerated ATP binds N-domain, energy opens gate
K+ exits
Actuator domain resets and cycle repeats
What is the Gibbs-Donnan effect?
Describes the unequal distribution of permeant ions on either side of membrane which occurs in presence of impermeant charged ions and large polar molecules
How does the Na+-K+ pump counter act the GD effect?
Negatively charged proteins hold onto positive ions in cell
With the dissolved solutes - low water potential
Na+/K+ -ATPase lowers number of dissolved particles inside the cell
How do secondary core transporters work?
Use kinetic energy provided by electrochemical gradients
Transport one solute down concentration gradient with transport of another solute against concentration gradient
How do symporters work?
They transport 2 or are molecules/ions in the same direction using integral membrane proteins
What are examples of a symporters?
Na+/glucose transporter (SGLT)
Na+/glucose transporter
Na+/phosphate transporter
Na+/K+/Cl- transporter
Na+/HCO3- transporter
Cl-/K+ transporter
What are antiporters?
They are integral membrane proteins that transport two or more
What are examples of antiporters?
Na+/H+ (NHE) - maintains cytosolic pH (stomach)
Na+/Ca2+ (NCX) - maintains low cytosolic [Ca2+] (cardiac muscle)
What does ABC transporters stand for?
ATP Binding Cassette transporters
What do ABC transporters transport?
Small molecules
What are the properties of ABC transporters?
Two membrane spanning domains
Two nucleotide spanning domains (NBDs)
R - regulatory domain (for function)
Presence of ATP in NBD and phosphorylation of R domain increases probability of opening
What is the CFTR transporter?
It is an example of an atypical ABC transporter
ATP regulates (influences opening) the channel rather than being a pure transporter
What is the difference between osmolarity and osmolality?
Osmolarity: total concentration of dissolved particles in a litre of solution
Osmolality: the number of dissolved particles per unit mass
What is osmotic pressure?
Pressure exerted by flow of water across membrane
Determined by solute concentrations
What is the gradient for water potential?
Low to high osmolarity
(hypo-osmolar to hyper-osmolar)
What is tonicity?
A measure of the effect a solution has on cells placed in it and is driven by osmolarity
What is the osmolarity of a hypo and hypertonic solutions in the context of cells?
Hypo: less than intracellular
Hyper: greater than intracellular
What is the equation for water transport?
Jv = Lp x ∆P
Jv - water flow (flux)
Lp - hydraulic water permeability
P - pressure
When do Lp and RT remain constant?
In a stable environment
What is a more complex equation for water transport involving RT, solute coefficient and concentration difference?
Jv = Lp x σ x RT x ∆C
Jv - water flow
Lp - hydraulic water permeability
σ - solute reflection coefficient
RT - gas constant and temperature
C - concentration
How can the 2 water transport equations be combined assuming solute coefficient is 1?
∆P = σ x RT x ∆P
What do the different values for solute coefficient (0-1) mean?
0 - fully permeable to membrane
0.5 - partially permeable
1 - semi-permeable to water
What is the structure of an aquaporin?
6 transmembrane domains
2 hemipores
Forms hourglass like structure for water transport
Where are epithelia found?
Line outside of the body
Line internal cavities and lumen of bodies
How are epithelia important in disease?
85% of human cancers originate from cells of epithelial origin
Cystic fibrosis is a disease of epithelial tissue
What are the different functions of epithelia?
Protection - covering + lining from external environment preventing desiccation (falling out)
Filtration - lining of kidney tubules
Exchange - alveoli
Absorption - intestine
Sensation - taste buds and olfactory epithelium
Secretion - lining of glands
What is the function and location of simple squamous type epithelia?
It is a fat single layer of cells
Function: absorption, filtration, minimal barrier to diffusion
Location: capillaries, alveoli, abdominal and pleural cavities
What is the function and location of simple cuboidal type epithelia?
Function: secretion (from opposite ends) and transportation
Location: glands, ducts, kidney tubules and ovaries
What is the function and location of simple columnar type epithelia?
Function: absorption, protection and secretion
Location: digestive tract
ciliated and non-ciliated
What is the function and location of stratified squamous type epithelia?
Function: protection
Locations: skin, mouth, upper throat and oesophagus
Keratinised (protected by keratin - impermeable and dry) or non-keratinised (kept moist prevents drying out)
Forms layers of cells
What is the function and location of pseudostratified columnar type epithelia?
Function: absorption and protection
Location: upper respiratory tract and trachea
Appear stratified due to position of nuclei
What is the function and location of transitional type epithelia?
Function: stretchable layer
Location: bladder
2 forms: relaxed or stretched
How do epithelia form a functional unit?
They are attached to the extracellular matriculates of the basal lamina
Cells are linked together using tight junctions, anchoring junctions and channel forming junctions
What are the properties of tight/occluding junctions?
Membrane proteins seal adjacent cells together
Ensure molecules cannot leak freely between cells
Prevent lateral migration of membrane proteins
How do tight junctions impact cell polarity?
They allow molecules to be transported apically and basally
Allows specific transportation of molecules
What does the paracellular barrier of tight junctions allow?
The control of the flow of molecules in the intracellular space
How are tight and leaky epithelia formed?
Claudin-1 and 3 used to bind epithelia tight
Claudin-1 and 2 form kinks in between epithelia which provides a leaky structure
What are anchoring junctions?
Used to provide mechanical stability
Anchor to basal lamina or other cells
Allows functioning as cohesive unit
How are anchoring junctions categorised?
Actin attached:
Cell-cell - adherent junction
Cell-basal lamina - focal adhesions
Intermediate filament attached:
Cell-cell - desmosomes
Cell-basal lamina - hemidesmosomes
What is the structure of the basal lamina?
40-120nm thick
Strong, flexible foundation - underlies epithelia
Meshwork formed by interactions
What are the different interactions of meshwork in basal lamina?
Laminin
Type IV collagen
Entactin
Perlecan
What is the basement membrane?
A combination of the basal lamina and reticular lamina (mainly type II collagen)
Main function is anchoring of epithelial ells to connective tissue
Acts as mechanical barrier
Important in angiogenesis
What are gap/channel forming junctions?
Allow diffusion between cells
Enable cell-cell communication
What is an ussing chamber?
A way to measure resistance of ion transport
Used to determine what ion channels are present
Uses short circuit current
What are the studies of frog skin using an Ussing chamber?
Used as model system
Absorbs Na+ from brackish (salty) water across skin
Skin dissected and mounted as flat sheet between two chambers containing solution of identical composition
Skins developed a transepithelilal potential difference due to active transepithelial Na+ movement
What is transepithelila voltage and how is it measured?
Polarised epithelial cells generate voltage/potential difference
Total difference in charge across cell
transepithelial voltage = Vte = (Vbl - Vap)
What are the key steps in epithelial NaCl and water absorption?
Passive facilitative entry of Na+ across apical membrane
Active exit of Na+ across the basolateral membrane
Paracellular diffusion of Cl- through tight junctions
Osmotically driven water absorption
What is the pump-leak model?
Mechanism of epithelial NaCl and fluid absorption in epithelial cells
How does the pump-leak model work?
One major type of Na+ influx channel - epithelial sodium channel (ENaC)
Na+ actively pumped out of cells across basolateral membrane via Na+/K+ ATPase
Causing paracellular transport of Cl- via tight junctions to maintain electroneutrality
Increasing NaCl concentration (lower WP) on basolateral side driving osmotic movement of water using AQPs or TJs
What is the function of K+ in the pump-leak model?
To maintain membrane potential and recycle K+
What is the role of ENaC in the kidney?
Na+ retention; control of whole-body Na+ and water balance hence blood pressure
Determines final urinary salt composition through regulatory action of hormones (aldosterone)
Long-term regulation of whole-body Na+ and water balance allows changes in ENaC function in aldosterone-sensitive distal nephron (ASDN)
What is the role of ENaC in the lung?
Na+ and water absorption; control of the amount of airway surface liquid (ASL) and alveolar lining fluid (ALF)
What is the role of ENaC in the colon?
Na+ and water reabsorption from the diet
What is the role of ENaC in the sweat gland?
Na+ retention; reabsorption of Na+ by sweat ducts, not followed by water
Creates hypotonic sweat solution
Which family is ENaC from?
Acid-sensing/degenerin ion channel family
What is the structure of ENaC?
Heterotrimer of 3 subunits coded by 3 genes
2 transmembrane domains form a pore
Long extracellular loops are site for regulation by CAPs and SLUNC1
PY-motif in C-term is site for ubiquination
What are the properties of ENaC?
Highly selective for Na+
Constituently activate - can be regulated
What is a specific inhibitor of ENaC?
Amiloride
What is the ASDN?
Aldosterone-sensitive distal nephron
What is the importance of the ASDN?
In long-term regulation of blood pressure
Where is the ASDN located?
In the last third of the distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD)
How is Na+ reabsorption stimulated in the kidney?
Through ENaC by principle cells in ASDN
What is the overall effect of ASDN?
Increased NaCl and H2O absorption, blood volume and blood pressure rises
How does aldosterone act on principle cells?
Aldosterone binds to cytosolic mineralocorticoid receptor (MR)
Goes to nucleus and binds to genes with HREs, levels of serum and glucocorticoid regulated kinase 1 (SGK) gene increase within 1 hour
Aldosterone increases
Surface ENaC levels by 2-5 fold
Na+/K+ ATPase density
ATP supply to support increased Na+/K+ ATPase activity
K+ excretion across apical membrane via ROMK
What happens in principle cells once aldosterone levels increase?
Surface ENaC levels by 2-5 fold
Na+/K+ ATPase density
ATP supply to support increased Na+/K+ ATPase activity
K+ excretion across apical membrane via ROMK (renal outer medullary potassium channel)
How does aldosterone stimulate Na+ reabsorption?
Increase in ENaC
How is the number of ENaC channels controlled?
By rate of insertion versus rate of retrieval/degredation
How is the retrieval and degradation of ENaC regulated?
By ubiquitin ligase (NEDD4-2)
How does NEDD4-2 regulate ENaC ubiquitination?
Binds to PY-motif of α, β or γ and adds ubiquitin group to a lysine residue in N-term of ENaC subunits
Ubiquitination ‘signals’ internalisation of ENaC followed by degredation
How is ENaC ubiquitination inhibited?
By aldosterone stimulating serum and glucocorticoid regulated kinase 1 (SGK1) within 1 hour
SGK1 phosphorylated NEDD4-2 which prevents binding to ENaC
So ENaC channels increase
What is hypertension caused by?
Dysregulation of ENaC
Excess aldosterone/mineralocorticoid action
OR
Liddle’s syndrome - gain function in ENaC
What is Liddle’s syndrome?
A rare autosomal dominant genetic disease caused by mutations in genes encoding 3 subunits (SCNN1A, SCNN1B and SCNN1G)
What happens in Liddle’s syndrome?
Changes in cytoplasmic regions of ENaC subunits which prevents NEDD4-2 binding and numbers of channels increase resulting in hypertension
Leads to low blood K+ levels (hypokalaemia)
How is K+ excretion enhanced in Liddle’s syndrome?
ENaC function depolarises apical mp
Electrical gradient for K+ excretion through ROMK is increased so excessive loss of K+ in urine so low blood K+
How can amiloride be used for Liddle’s syndrome?
Inhibits ENaC and hyper polarises apical mp reducing K+ loss helping prevent hypokalaemia and lowering blood pressure
Where is ENaC expressed in the lungs?
In the apical membrane of surface epithelial and alveolar type II cells
What does ENaC regulate in the lungs?
The airway surface liquid (ASL) in conducting airways which is crucial for innate defence via mucocillary clearance
AND
Alveolar lung fluid (ALF) which is essential for efficient gas transfer
How do channel-activating proteases regulate ENaC?
By increasing ENaC activity via cleavage of external loops of α and γ ENaC
How do anti-proteases regulate ENaC?
They reduce ENaC activity by inhibiting these channel-activating proteases (e.g. aprotinin)
How does SPLUNC1 regulate ENaC?
By reducing ENaC function
Secreted into ASL and binds to ENaC which protects from internalisation and CAP activation
How do other local acting factors regulate ENaC?
They are secreted into ASL (e.g. ATP)
What is the experimental evidence of ENaC regulation using proteases?
Cells cultured and isolated and expanded
Differentiation at air-liquid interface form pseudo stratified single layer of epithelial cells
Tubulin - ciliated cells
MUC5AC - goblet cells
What is the role of the NKCC1 co-transporter?
In 2 types of Cl- channels: CFTR and Ca2+ activated Cl- channel (CaCC)
What is the mechanism of NaCl secretion via epithelial cells in the GI tract, exocrine glands and conducting airways?
Na+-K+-2Cl- transports Cl- into the cell and Cl- exit via CaCC
K+ into cell through transporter and Na+/K+ pump and transported out due to electrical gradient
What are the 5 major domains of CFTR?
MSD1 & 2 - form pore of channel
NBD1 & 2 - bind ATP
RD - site of PKA phosphorylation
What is the mechanism of CFTR gating?
PKA phosphorylation of RD induces ATP binding and dimerisation of NBDs
Conformational change in NBDs transmitted to MSDs pore opens
ATP hydrolysed and pore closes (if another ATP binds channel re-opens as long as RD phosphorylated)
Dephosphorylation by protein phosphotases closes channel
What are the requirements for CFTR to open?
PKA phosphorylation and ATP binding
What is the evidence for the use of PKA phosphorylation and ATP binding for CFTR?
Patch-clamp technique using an inside-out patch to monitor the channels and the requirements to access the channel
Where are calcium-activated Cl- channels found?
Int he apical membrane of most epithelial cells that express CFTR (NOT intestinal)
Gland secretory acing cells (no CFTR)
How are CaCC activated?
By a rise in cytosolic Ca2+
Generally transient
Which family are CaCCs apart of?
TMEM16 (A & B)
What can be used to regulate CaCC?
Calmodulin (CaM) and CAM dependent kinase (CaMK)
Not essential though!
What is the structure of TMEM16A?
10 transmembrane domains - pore is domains 6-9
Crystal structures indicate functional channel is a dimer
How is TMEM16A activated?
Ca2+ bind to glutamate residues in one of the 2 α helices of intracellular loop 3 (ICL3), α helices move apart open pore enabling Cl- transport
Closes when cytosolic Ca2+ is reduced back to resting levels
What are the 2 mains mechanisms of HCO3- secretion?
Directly through Cl- channel
Indirectly through coupling Cl- channel with apical Cl-/HCO3- exchanges
CFTR uses both modes
What is the SLC26 family?
Transporters anion exchanges belong to
What does SLC stand for?
Solute carriers
What is the net result of Cl- channel and Cl-/HCO3- anion exchanger coupling?
Net epithelial NaHCO3 secretion
When are Na+ dependent HCO3- cotransporters involved?
They are located on the basolateral membrane that supplies cytosolic HCO3- for the exchanger (SLC4 family)
How does CFTR regulate SLC26A anion exchanger in epithelial cells?
PKA phosphorylation switches on AE activity for SLC26, A3, A6 and A9
Required physical interaction of 2 proteins aided by scaffold protein CAP70 and CFTR RD phosphorylation
What happens to SLC is CFTR is dysfunctional?
AE is inhibited deducting net HCO3- and fluid secretion
Where is HCO3- secretion regulated by CFTR?
Small intestine
Biliary tract
Exocrine pancreas
Airway
Female and male reproductive tracts
How does anion and fluid secretion take place in the acinar cells?
Secrete vary of digestive enzymes and low volume, NaCL-rich fluid into ducts using TMEM16A channels
How does anion and fluid secretion take place in duct cells?
Transport digestive enzymes to small intestine
Produce high volume NAHCO3 rich secretion using CFTR and SLC26A6
Why is HCO3- essential in exocrine pancreas?
They solubilise the protein allowing transport down to small intestive
Also HCO3- is needed for the digestive enzymes
What happens if fluid absorption fails or if there is excessive fluid secretion?
Leads to rapid dehydration, electrolyte imbalance and even death
How is fluid secretion and absorption driven in GI tract?
Secretion by anion (Cl- and HCO3-) secretion
Absorption by secondary to sodium absorption and involves multiple mechanisms
Approximately how much liquid is secreted and absorbed in the GI tract?
Food and saliva deliver ~3000ml
Stomach - secretes: 2000ml
Duodenum - secretes: 1000ml (pancreas) and 400ml (bile)
Small intestine - secretes: 2600ml and absorbs: 7900ml
Large intestine - absorbs: 1000ml
Net loss ~100ml
Where does ENaC-mediated fluid absorption occur?
In the colon
How does electroneutral NaCl absorption take place in GI?
Using sodium-linked absorptive ion transporters (SLC26A6 (PAT1), NHE3 and SLC36A3 (DRA))
What happens when mutations occur in the DRA transporter?
Cl- losing diarrhoea
How are different nutrients absorbed?
Nutrient absorptive transporters (PAT1, B0AT1, SLGT1, GLUT5 and PEPT1)
What are the main causes of secretory diarrhoea?
Due to dysregulation in cell signalling
How can secretory diarrhoea be prevented?
Through safe drinking-water and adequate sanitation and hygiene
How does vibrio cholera cause secretory diarrhoea?
Cholera toxin inhibits NaCl and fluid absorption
Stimulates CFTR-mediated Cl-/HCO3- and fluid secretion
What are the covalent modifications caused by cholera toxin?
Modifies Gα subunit
ADP-ribosylation of G-proteins blocking GTP hydrolysis
AC becomes permanently active
What does the covalent modification of the g-protein lead to?
Uncontrolled, over-stimulation of CFTR and inhibition of electroneutral NaCl absorption
Excessive salt and water loss into intestinal lumen producing severe diarrhoea
What is the treatment of secretory diarrhoea?
Oral rehydration therapy or intravenous fluid
What is oral rehydration therapy?
Isomolar salt solution containing NaCl, NaCitrate, KCl and glucose
Starch used to prolong treatment
Causes rapid fluid reabsorption utilising nutrient absorptive transporters
When was the first cystic fibrosis gene cloned?
1989
What is cystic fibrosis?
Autosomal recessive disease (mostly in caucasians)
Loss of function in CFTR gene (regulates volume and pH)
How is cystic fibrosis characterised?
By a severe lung and pancreatic disease, also includes GI and reproductive tracts, sweat glands and kidneys
How much morbidity and mortality does CF lung disease count for? (%)
> 90%
Approximately how many people have CF in the UK?
> 11000
How many people in the UK are carriers of CF?
1 person in ~25
What was the life expectancy and what is it now?
1964 - 5 years
Now - >50
How many mutations of CFTR exist?
2000
What is the most common mutation?
F508del - loss of phenylalanine at position 508 NBD1
What are the different classes of function cystic fibrosis mutations?
Class I - No protein (G5A2X)
Class II - No traffic: degraded proteases (F508del)
Class II - No function: e.g. doesn’t respond to PKA (G551D)
Class IV - Less function: open probability, low permeability (R117H)
Class V - Less protein (A455E)
Class VI - Less stable: doesn’t have normal half-life (rF508del)
What are the different types of cystic fibrosis mutation?
Class I-III = minimal to no function mutations
Class IV-VI = residual function mutations
How can phenotype of CF help predict genotype?
Tests in different organs, suggests severity of ion transporters
Lung CF can cause different major problems
(severe usually means sweat glands and pancreas are bad at functioning)
What is the difference between a normal and diseased exocrine pancreas?
Normal
NaHCO3-rich alkaline fluid secretion (Duct cells)
NaCl-rich acidic secretion (acinar cells)
Diseased
No HCO3- in duct so no alkaline environment
Acidic solution (NaCl) released which creates a blockage so acinar cells destroyed
What treatment is required to allow pancreas function in CF?
Pancreatic enzyme replacement therapy (PERT) required at every meal
What are the different cells in the conducting airways containing CFTR?
Club cells
Goblet cells
Basal cells
Ciliated cells
Ionocytes
PNEC
Tuft cell
Which cells have the most CFTR in the conducting airways?
Club cells (most important for secretion)
Goblet cell
Basal cells
Ionocyte
What is the function of the goblet cell in the conducting airways?
Exocytosis of the granules and mucins (used in mucosal layer of ASL)
What is the role of CFTR in conducting airways?
Maintains proper hydration and pH of ASL, including PCL and mucus layer
Ensures efficient mucociliary clearance (MCC) which removes pathogens that get trapped in mucus layer
Surface cells and submucosal glands also secrete antimicrobial peptides into ASL
Where are submucosal glands located?
In the larger airways
What happens when mucociliary clearance fails?
Leads to lung disease in CF
How does mucociliary clearance fail?
As the ASL is dehydrated and more acidic which leads to viscous mucus, mucus accumulation and obstruction and failure in MCC
Chronic bacterial colonisation (biofilm forms), airway inflammation and respiratory failure
How is pH more acidic in ASL in CF?
Lack of function of CFTR reduces HCO3- secretion in ASL
With active H+ secretion from airway cells more acidic pH is produced
What are the consequences of a more acidic ASL?
Increased fluid absorption due to enhances ENaC activity - increased channel activating proteases (CAP) activity and decreased SPLUNC1
Increased mucus stasis and viscosity - decreased mucin release and expansion on release from goblet cells and conformational changes in in mucins making them more rigid
Decreased bacterial killing - reduced AMP activity
How do defects in CFTR cause lung disease?
Decreased Cl- and HCO3- transport
Decreased ASL volume and pH
Increased ENaC function
Increased mucus obstruction
Decreased MCC
Decreased antimicrobial activity
Increased bacterial and viral infections
Increased inflammation
Destruction of lung
How can mucus clogging be treated in CF?
Using physiotherapy and mcolytics
How can ASL be restored in CF?
Nebulised hypertonic saline or mannitol
draws water from body into airways improving hydration - transient effect
How can recurrent lung infections be treated in CF?
Using antibiotics
How can overactive immune response be treated in CF?
NSAIDs
How can destruction of the lung be treated in CF?
It cannot a lung transplant is required
What are the different approaches to treat basic anion permeability defect in CF?
CFTR modulator therapy
Genetic therapy
Alternative channel therapy
How much CFTR function does a CF patient have?
5% - increased sweat chloride and lung disease
1% pancreatic disease
How can therapies be assessed in vivo?
Lung function (e.g. FEV1.0)
Sweat chloride (sweat test)
Other parameters (Number hospitalisations, BMI and quality of life)
How is CFTR involved in sweat secretion and reabsorption?
In the absorptive duct transcellular NaCl absorption produces a hypotonic fluid
In which CFTR and ENaC are both involved in the transcellular absorption of NaCl
How is calcium signalling involved in sweat production?
Uses TMEM16A transporter of Cl- which creates a NaCl rise isotonic secretion from the secretory coil of the sweat gland
How are ions and fluid transported in a normal sweat gland?
ENaC and CFTR are used to transport NaCl for salt reabsorption
How are sweat glands regulated?
Mostly via Ach as well as catecholamines
How are ions and fluid transported in a CF sweat gland?
NaCl is not reabsorbed as there is no CFTR function so sweat becomes very salty
What are the normal sweat salt concentrations?
Below 60mM
What are the sweat salt concentrations in cystic fibrosis?
It can be as high as 120mM
What is CFTR modular therapy?
The use of chemicals to correct the mutant CFTR
Used alone or in combination with genetic or alternate channel therapy
Depends on mutation
Personalised therapy may be required
What aspects of CFTR mutants can be altered using drug therapy?
Increasing the number of CFTR channels at cell surface (Class II - F508del)
Enhance CFTR channel gating (Po) (Class III - G551D)
Increase ion flux (Class IV - R117H)
Which company first founded CFTR modular therapy?
Vertex pharmaceuticals
What are the different types of CFTR modulators?
Potentiators
Correctors
Termination suppressors
Amplifiers
Stabilisers
What are the CFTR modulators in clinical use?
Potentiators and correctors
What are potentiators?
They are used to increase the activity of CFTR of class III (G551D) gating mutations and some RF mutations
How do potentiators work?
They increase opening rate or duration of opening (channels have to be phosphorylated)
What are the different potentiators in clinical use?
Vertex VX-770 Kalydeco (US), Ivacaftor (UK)
How does the VX-770 potentiator work?
Increased the open probability via an ATP-independent mechanism
When did FDA approve Kalydeco?
Jan 2012
When was Ivacaftor approved in the UK?
Aug/Dec 2012
How many mutants is Ivacaftor used for currently?
97
What are correctors?
Chemicals that promote processing of class II (F508del) mutants to the plasma membrane
How do correctors work?
They improve the processing of the mutant from the ER to the Golgi by increasing folding efficiency of the channel in the ER
Then escapes ER quality control (ERQC) machine’s meaning it is not degraded
Increasing number of channels
What is the corrector in clinical use?
VX-809 Iumacaftor
How were correctors use investigated using Ussing chambers?
Chloride secretion was measured after stimulation with cAMP agonist after 48 hours treatment of treatment
Why did patients homozygous for F508del show little effect on lung function when treated with VX-809?
Because there are multiple defects that have to be treated
What are the 3 defects that have to be corrected in F508del CFTR?
Processing defect (low N)
Gating effect (lower Po than WT)
Rescued F508del CFTR has shorter resident time (stability) in plasma membrane
How can the problem using Iumacaftor only on F508del be solved?
By using combination therapies using VX-770 as well as VC-809
When did FDA approve the F508del combination therapy?
In 2015 (Orkambi)
When was the triple therapy approved?
In 2018
corrector VX-445(elexacaftor) + VX-661 (tezacaftor) + VX-770 (ivacaftor)
Who could use the triple therapy?
F508del/minimal function and F508del/F508del patients
What are the highly effective modulator therapies in use?
Ivacaftor, Orkambi, Symkevi and Kaftrio
What are the different CFTR modulators under development?
Termination suppressors
Amplifiers
Stabilisers
How do read through agents work in CFTR?
They are used for class I CFTR mutations (G542X) stopping the premature degradation of the protein
What are the different modulators for class V?
Amplifiers - creating more CFTR in the membrane, increasing expression
What are the different modulators for class VI?
Stabilisers stopping the CFTR channel from leaving the membrane
Approximately what percentage of people benefit from the triple therapy for CF?
F508del//F508del (40%)
F508del//other (34%)
Approximately how many people benefit from potentiators?
G542D +9 gating +23 residual function mutations (~8%)
How many patients with CF have no drug treatment?
~18%
How does the gene therapy used for CF work?
Copies of healthy gene are delivered using gene addition
Chromosomal DNA can be fixed using genome editing
What were the main problems for gene therapy for CF?
The physical and/or immune barriers
When viral vectors were used viral proteins were synthesised which meant cells were destroyed
How can gene therapy be improved?
Using different gene transfer agents
e.g. Lentivirus - long term and stable from a single dose because gene is integrated into DNA
Virus modified to make less immunogenicity
Nano particles have also been used
What are the problems with gene therapy?
Correct cells need to be targeted (best = basal (stem) cells)
Mucus important barrier to gene therapy
Potential for distraction of other genes
What are other genetic and cell based approaches?
Gene editing therapies
mRNA-mediated therapies
Antisense Oligonucleotide (ASO)-mediated therapy
What is alternate channel therapy?
It is an independent CF mutation therapy
Uses alternative chloride channels (ACCs) that are present in CF cells to bypass CFTR and restore Cl-/HCO3-
Use inhibitors of ENaC to help reduce salt and fluid absorption
What types of drugs can be used to target ENaC activity?
Amiloride-like drugs (inhibitors)
Target ENaC regulation (inhibit proteases, target ENaC regulatory proteins)
What are other approaches to CF therapies?
Using synthetic anion channels and transporters
What are the 2 main transporter superfamilies?
ATP-binding cassette (ABC) superfamily
Solute carrier (SLC) superfamily
What are the different subfamilies in solute carrier superfamily?
OAT - organic anion transporter
OATP - organic anion transporting polypeptide
OCT - organic cation transporter
MATE - multidrug and toxin extrusion protein
What are ABC transporters?
Membrane proteins that couple substrate transport to ATP hydrolysis
Importers OR exporters
What are the ABC exporters?
ABCB1/MDR1/P-gp
What are the ABC importers?
Found in prokaryotes:
Maltose uptake transporter
Methionine uptake transporter
What are the different associated ABC transporters?
CFTR (ABCC7)
SUR1 (ABCC8)
Where are the ABC transporters expressed?
Liver
Intestine
Kidney
BBB
Blood placenta barrier
Blood testis barrier
Where in cells will ABC transporters be expressed?
On the apical or basolateral side (not both)
What are ABC transporters protective function?
To protect the body against xenobiotics (toxic chemicals)
What is an example of a normal physiological role of ABC transporters?
Bile acid transport in liver or regulation of insulin release in pancreas
How many genes do ABC transporters have?
48
What are the domains in ABC transporters?
4 domains
2 nucleotide binding domains (NBD)(hydrolyse ATP)
2 transmembrane domains (TMD) bind and transport substrates
What are the properties of the NBD and TMDs?
NBDs - highly conserved
TMDs - less conserved
What are the properties of ABC half transporters?
1 TMD and NBD
Homo or heterodimers
What are ABC transporter roles in the intestine?
Drugs enter enterocytes through brush border membrane and cross basolateral membrane into hepatic portal vein to circulate
ABC transporters interfere with this process and pump compounds like drugs into the lumen
What are ABC transporter roles in the liver?
Drugs transported into hepatocyte from circulation across sinusoidal (endothelium) membrane
Drugs are transported out either across canalicular membrane (bile) or sinusoidal membrane (blood/renal excretion)
What are ABC transporter roles in the BBB?
Prevent potentially toxic compounds reaching brain
KO mice studies show drug accumulation in brain and toxicity
How do cancer cells show multi drug resistance?
Decreased uptake
Increased metabolism of drug
Alteration in cell target
Enhanced drug efflux
Where does enhanced drug efflux take place?
Takes place in MDR transporters
MDR1, MRP1 and ABCG2
What is P-glycoprotein (ABCB1)?
It is a main multi drug resistant transporter (mainly in cancer cells)
Also called MDR1 (multi drug resistant)
Present in normal tissues and many tumour cell lines
ABCB1 gene on C7 codes for protein
What is the structure of PGP?
12α helices
2 TMDs
2 NBDs in cytoplasm
What is the proposed model of PGP transport?
TMD and NBD are both fair apart when unbound
ATP binds to NBD and substrate binds to binding site
ATP hydrolysis occurs and conformational change takes place (NBDs come together) so drug effluxes
ADP is released and ATP is hydrolysed resetting the transporter
Process repeats
What is the role of PGP?
Removal of xenobiotics
Pumps them from enterocytes following initial absorption
Transports xenobiotics into bile across canalicular membrane
Prevents access of drugs to brain
Transports drugs to lumen of kidney on brush-border membrane
When do cancer cells express PGP?
After treatment causing drug resistance
Where is PGP expressed?
Liver
Kidney
Intestine
BBB
Blood placenta barrier
Blood testis barrier
What are the different effects of PGP in tissues?
Limited drug absorption (gut lumen)
Active drug elimination (PCT and hepatocytes)
Limited drug distribution in tissues (endothelial cells and (synctiotrophoblast) fetal tissues/capillaries)
What were the PGP knockout studies in mice?
Viable, fertile and phenotypically normal
Suffer toxicity with some compounds due to entry into brain - once gets into brain most likely die
Increased absorption and decreased excretion of different drugs
How are PGP and CYP3A4 related?
Substrate specificity of Pgp overlaps with CYP3A4
Pgp induced through PXR (xenobiotic sensor) receptor by compounds such as rifampicin
How does St Johns wort impact both Pgp and CYP3A4?
Herbal remedy for low mood and mild anxiety
Increases Pgp expression and hence efflux activity
Induces CYP3A4
How does p53 and Pgp impact drug resistance?
p53 is a tumour suppressor gene
Inactivates in ~50% of cancers
Associated with drug resistance and poor prognosis
Wild type p53 represses Pgp transcription via direct DNA binding and down regulates Pgp via mrR-34a and LRPPRC (protein coding gene)
Mutant p53 cooperates with ETS-1 (TF) up regulating ABCB1 expression
How does Pgp help drug resistance using lysosomal sequestration?
Lysosomal accumulation of anticancer drugs as novel mechanism
Plasma membrane with Pgp buds inwards forming early endoscopes
Endosomes to mature lysosome
Drugs enter cell and lysosome
Becomes charged and trapped
What is BSEP?
Bile Salt Export Pump
What is the function of BSEP?
Transports bile salts across canalicular membrane of hepatocyte
Transports some drugs (vinblastine)
What disease is associated with a defect in BSEP?
PFIC2 (progressive familial intrahepatic cholestasis)
Blocks secretion of bile
What is ABCB4?
A specific translocate for phosphatidylcholine
Translocates phosphatidylcholine from inner to outer leaflet of canalicular membrane for extraction
Form bile salts protects hepatocyte biliary membrane
What happens in mutations of ABCB4?
3 different hepatobiliary diseases
Progressive familial intrahepatic cholestasis type 3
Gallstones and intrahepatic cholestasis of pregnancy
What types of chemotherapies does ABCB4 transport efflux?
Anthracyclines
Texanes
Vinca alkaloids
What are the MRP transporters?
Multi-drug resistance-associated protein (MRP)/ABCC
ATP-dependent high MW membrane proteins
12 different known
Variety of functions, protection from xenobiotics to channeling ions
Facilitate the extrusion of numerous glutathione, glucuronate and sulphate conjugates
Expressed in numerous tissues (ubiquitous)
How does the structure of MRP1 differ to Pgp?
MRP1 has an additional transmembrane domain (5 extra α helical regions)
What are the properties of MRP1?
ABCC1 gene on chromosome 16
190 kDa
Main contributor for multi drug resistance
Expressed in high levels in a variety of tissues, low levels in the liver
Preference for amphiphilic organic anions
What is LTC4?
Leukotriene C4
What is LTC4 used for?
It is a physiological high affinity substrate
Family of lipid mediators of inflammation synthesised from arachidonic acid
MRP1 mediates transport of LTC4 across PM
Formation in lung important in asthma and allergy
How is LTC4 formed?
By conjugation of GSH to LTA4 through reaction catalysed by leukotriene synthase enzyme, active in eosinophils, monocytes neutrophils and macrophages
How was the function of LTC4 found?
Using knockouts in mice which showed an inflammatory response
What is the phase II metabolism of drugs?
Conjugation reactions with glutathione
How do MRP1 and glutathione interact?
Can transport glutathione conjugates by using GSH as a cotransporter
What happens to mice when MRP1 is knocked out?
No significant difference in viability or fertility
Elevated GSH
Unchanged tissue levels of GSH in organs expressing little/no MRP1 (liver + small intestine)
Dispensable is dispensable for development and growth
Increased sensitivity to several chemotherapies
What is the oncogenic regulator of MRP1?
It is a downstream target of MYCN in neuroblastoma
What are the MYCN and ABC transporters?
MYCN amplification and MDR1 overexpression are frequently observed
MDR1 is a target of MYCN
MYCN directly regulates expression of MRP1
MYCN regulates expression of a range of ABC transporters
How does MRP2 transport glucoronides?
High level of expression on bile canaliculus of the hepatocyte (on apical membranes of kidney and intestine)
Important contribution to elimination of drug glucuronides in bile (diclofenac, morphine and fexofenadine)
Important physiological role in elimination of bilirubin from the body
How do Haem metabolism and MRP2 interact?
MRP2 (ABCC2) transports bilirubin glucuronide from liver to bile
In rare metabolic disease Dubin-Johnson syndrome no active MRP2 due to mutations
Individuals have high levels of bilirubin glucuronide in their plasma
Usually a benign condition may see jaundice in pregnancy or with some drugs
What is ABCC8?
Also known as SUR1
Sulphonylurea receptor used in control of blood glucose
No transport role but ATP-sensitive regulator of potassium channel
Sulfonylureas bind to receptors causing effect on K+ channel
Membrane potential more positive opening VG Ca2+ channels
Rise in calcium increased insulin secretion
What is ABCG2/BCRP?
BCRP (Breast cancer-related protein) first identified in breast cancer line
Expressed in other tissues and relevant to drug excretion
MXR (mitoxantrone-resistance protein)
What is the structure of ABCG2?
Single spanning transporters
Smaller than ABCB and ABCC
Half-transporter
~70 kDa
Form homodimers
Where is ABCG2 located?
Similar to Pgp
High levels in lactating breast
Secretion of xenobiotic into milk which has implications for breast-fed infants
Restrictions on use of certain drugs by nursing mothers
What does ABCG2 have the most implications on?
Secretion of xenobiotic into milk which has implications for breast-fed infants
Restrictions on use of certain drugs by nursing mothers
What effect does ABCG2 have on cancer?
Upregulation in CML-initiating cells so resistance to certain chemotherapies (Imatinib)
How does ABCG2 cause Imatinib resistance?
Evidence that it is a substrate for the transporter
CML stem cells have higher levels ABCG2 than mature ones (less sensitive to chemo)
May involve decreased levels of regulatory miRNA miR-212 resulting in +ABCG2 levels
Imatinib-mediated inhibition of BCR-ABL - down regulated ABCG2 levels via PI3K-Akt pathway
What types of drugs can help stop certain cancers effluxing chemo?
TKIs to inhibit RTKs (growth factor signalling)
Non-toxic pathway, more specific
Acquired resistance is a problem
Important considerations with combination therapies
What is the link between ABCG2 and gout?
Genetic polymorphism resulting in point mutation in ABCG2 - risk of gout
Uric acid is a substrate for ABCG2 and unstable protein results in poor ability to excrete the protein
What is gout?
An accumulation of uric acid crystals in joints occurs resulting in pain and inflammation
uric acid is accumulation of purine metabolism
How is gout bad?
Cause effects in joints and kidneys (kidney stones)
What strategies are used to overcome ABC mediated multi-drug resistance?
Chemical inhibitors
Natural compounds
Antibodies
Reduced expression
Agents that bypass transporters
Novel delivery systems
What are the different generations of MDR I inhibitors?
1st:
Verapamil, quinidine, amidarone and cyclosporine A
2nd:
Valspodar and dexverpamil
3rd:
Dofequindar, zosuqudar,tariquidar, elacridar, biricodar
What is collateral sensitivity?
When chemotherapy efflux is blocked to chemo accumulates and GSH efflux is stimulated so GSH depletes
