Epithelial Chloride Channels 3&4

Question 1

What did rommens et al discover regarding CF 1989

Answer 1

Identification of CF gene

Question 2

What did Riordan et al in 1989 discover regarding CF

Answer 2

cDNA of CF gene

Question 3

What did Kerem et al in 1989 discover regarding CF

Answer 3

Genetic analysis of CF gene

Question 4

Where is the CF gene found

Answer 4

Human chromosome 7

Question 5

How long is the CF gene

Answer 5

250,000 base pairs of DNA

Question 6

What is the CF gene protein called

Answer 6

Cystic fibrosis transmemrbrane conductance regulator

Question 7

What is the commonest CF mutation

Answer 7

Fetoalanine deletion mutation at 508 in 70% of CF on chromosome 7

Question 8

What 4 epithelial tissues are affected by CF

Answer 8

Skin
Lung
Gut
Pancreas

Question 9

What is the CFTR a family of and why was this a surprise

Answer 9

ATP binding cassette family of transport protein (structural homology of this protein type) - surprise as mostly found in bacteria

This family use ATP to pump ions out of the cell.

Question 10

What is the structure of CFTR

Answer 10

2 hydrophobic membrane spanning domains (anchors to cell membrane) - comprised of 6 alpha helixes
2 nucleotide binding domains - bind and hydrolyse ATP (NBD1 is where the fenylalanine 508 is missing in CF patients)

Question 11

What is the unique Regulatory domain role

Answer 11

The phosphorylation site for PKA for the CFTR

Question 12

What did Rich et al 1990 discover regarding CF

Answer 12

Changing CF CFTR gene with normal one corrects defective chloride channel regulation in CF airway epithelial cells.

Also transfected normal CFTR with F508 CFTR and it did not induce appearance of CI currents

Question 13

What were the Cl channel type seen by Rich et al 1990

Answer 13

Channels induced by CFTR expression very similar to SLCCs not ORCCs.

Question 14

What actually is CFTR

Answer 14

SLCC - CFTR is a low conductance cAMP-activated chloride channel.

Question 15

How was CFTR confirmed to be SLCC by bear et al 1992

Answer 15

Used S9 insect line which is a cell containing no channels. They then added CFTR mRNA to make the CFTR protein purified.

CFTR is mixed with phsopholipds to get them into phospholipid vesicles.

This is then rubbed on a plate to form a phospholipid bilayer.

This allows CFTR to sit in the phospholipd bilayer where currents can take place to asses the channel characteristics.

Found cAMP activated SLCCs - final proof than CFTR is a low conductance cAMP-activated chloride channel.

Question 16

What did Kartner et al discover regarding CFTR in sweat glands in 1992

Answer 16

F508 CFTR was not found in apical membrane - first indication that CF may be due to absence of Cl channels rather than defects in them

Question 17

What did Denning et al in 1992 find out regarding CFTR in lung epithelium

Answer 17

Not present, backing kartner et al.

Also found that by lowering the temp that it allows F508 mutated CFTR to locate to the apical membrane and it also retained some function. Therefore - DeltaF508 mutation causes trafficking issue not a functional problem

Question 18

What did sheppard et al in 1993 find out regarding CFTR

Answer 18

Identification of 3 membrane spanning domains which effect arginine residues, as arginine is positive, this results in a reduced currents going through the cAMP-activated SLCC channels.

Therefore - arginine residues possibly determine Cl conductivity of channel

Question 19

What was found by Cotten and welsh in 1999 regarding CFTR pore architecture

Answer 19

They stated that the pore architectures is based on cationic and anionic amino acid residues - when swapping arginine (+) for aspartate (-) then it destabilises the channel and stops it working. But when swapped both the arg for asp at both sides it maintains the channel.

Question 20

What are the 6 sections of CFTR

Answer 20

2 - membrane spanning domains (MSD1 and 2)
2 - nucleotide binding domains (NBD1 and NBD2)
R domain
Glycosilation - quality control passed via golgi apparatus and ready to be used.

Question 21

What activates the CFTR

Answer 21

ATP on both NBD
PKA on the R domain

Question 22

What are the 4 areas that the CFTR can be affected causing CF

Answer 22

Genetic defect - protein production
Processing in golgi apparatus
Regulation - ATP or PKA issue
Conduction - issue in getting chloride through channel (e.g. channel architecture)

Question 23

Why is CF difficult to do studies on

Answer 23

No natural animal model
Mice have calcium activated Cl channels in lungs so they take on CFTR role when knocked out so the KO mice area symptomatic

Question 24

Who made the first CFTR KO mouse

Answer 24

Koller 1992

Question 25

What did Kollers CFTR KO mouse show

Answer 25

GI obstructions - and failure to thrive.
Only living about 40 days

Question 26

What did Gabriel et al discover regarding ORCCs and CFTR in 1993

Answer 26

ORCC activation only happens by PKA when CFTR is present in cells

Question 27

What did Jovov et al discover with regards to ORCC and CFTR with the mutant G551D (5% of CF)

Answer 27

Went put into lipid bilayers it also showed that functional CFTR expression is required for ORCC activation by PKA and ATP

Therefore - CFTR is needed to regulate ORCCs

Question 28

So in summary what is the role of the CFTR

Answer 28

Small linear conductance chloride channel whos added role is to regulate the outward regulating chloride channels.

Question 29

What is the commonest CF infection

Answer 29

Pseudomonas aeruginosa

Question 30

What happens to Pseudomonas aeruginosa in normal lungs

Answer 30

They die quickly

Question 31

What happens to Pseudomonas aeruginosa in CF lungs

Answer 31

They thrive - found by smith er al 1996

Question 32

What did Singh et al find regarding CF and bacteria in 1998

Answer 32

The body has epithelial beta defensins (HBD-1 and 2) were identified as natural bacterial agents.
They are found in airway surface liquid and kill in conc-dependent manner.
Dependent effectiveness on ionic strength of medium - higher the NaCl the less bacteria killed/less effective HBD1/2
Lysozyme - also affected by salt levels.

Question 33

What did conner et al in 2006 find out as to why CF patients are prone to infection

Answer 33

In the lung epithelial cells, there is a sodium thiocyanate transporter on the basomembrane side which brings sodium and thiocyanate into lumen cells.

Thiocyanate then leaves the cell into the lumen via the CFTR. w

hydrogen perioxide (made by duox enzyme) binds with lactoperoxidase in the lumen.

The intermediate compound of lactoperoxidase and H2O2 combine with thiocyanate to form hypothiocyanite.

Hypothiocyanaite is an effective bacteriocidal agent and kills off bacteria.

As CF patient doesnt have CFTR then no thiocyanate can get through to make the hypothiocyanite so no killing of bacteria.

Question 34

What are read-through compounds and how do they help for CF

Answer 34

Ensure the correct and complete genetic coding for the CFTR - E.g. - gentamicin

Question 35

How can CFTR chaperones help to treat CF

Answer 35

Stabilise protein structure/promote folding –> expression in apical membrane

Question 36

How can CFTR activators help to treat CF

Answer 36

Phosphoesterase inhibitors so stop cAMP from being broken down. E.g. - alkylxanthines

Question 37

How can CFTR potentiators help to treat CF

Answer 37

Correct and activate the channel e.g. VX-770

Question 38

How can MPB-07 work to treat CF

Answer 38

Activates CFTR the same way as cAMP - Becq et al 1999

Question 39

Why doesnt CFTR activators (e.g. IBMX) not work for the CFTR deltaF508

Answer 39

No CFTR to activate with the prolonged cAMP

Question 40

How can IBMX and MPB-07 work together to treat CF

Answer 40

MPB-07 activates CFTR like cAMP and allows it to get into the membrane for IBMX to allow cAMP to work for longer increasing the Cl functionality.

Question 41

What did Van Goor et al discover in 2009

Answer 41

Used iodide (as tracer) for CFTR channels and showed that VX-770/invacaftor is more effective at inducing current than FSK (natural cAMP activator) in CF patients with the G551D mutation, also showed small but significant increases in the F508delta mutation.

Question 42

What is orkambi

Answer 42

Ivacaftor combined with lumacaftor (chaperone for ivacaftor) - licensed in UK but very expensive

Question 43

What role does CFTR have on epithelial sodium channels (ENac) in the lungs

Answer 43

CFTR inhibits ENaC. So in CF where CFTR isnt present, this causes an increase in ENaC activity, which increases sodium reabsorption from the lumen which decreases the airway surface further via osmosis pulling the water back into the cells.

Question 44

What role does CFTR have on epithelial sodium channels (ENac) in the sweat glands

Answer 44

CFTR activates ENac in sweat glands so when CFTR is absent this causes inactivation of the ENaC which reduces salt reabsorption. Forming salty sweat.

Question 45

What other transporters are likely regulated by CFTR

Answer 45

C-terminus of CFTR binds to PDZ proteins which are important for regulation of ENaC, ORCC, ROMK2, AQP3

Question 46

What did middleton et al in 2019 show regarding CF treatment for those with the delta F508 mutation

Answer 46

Combo of elexacaftor-tezacaftor-ivacaftor showed a 13.6% increase in FEV1 values.

Also reduced exacerbation, decreased sweat chloride levels to normal, increased BMI and improved patient QoL.

Question 47

What effect does poor pancreatic function have on lung function in CF and how should this be treated

WIDER READING - Konstan et al 2003

Answer 47

Another study reported that growth indices, including weight-for-age and height-for-age, and symptoms of lung disease are highly associated with decreased lung function at age 6 years, and concluded that early treatment with pancreatic enzyme therapy and appropriate nutrition could improve lung health overall

Question 48

What are class I CF mutations

WIDER READING - Welsh and smith 1993

Answer 48

Mutations that result in defective protein production.
This is caused by premature termination signals, frameshift mutations (insertion, deletion or frameshift mutations)
These mRNAs are usually unstable and produce no protein however if they dothey are often degraded rapidly and do not produce functioning CFTR
No detected defective protein has been detected in cells.

Question 49

What class of mutations are the delta F508 mutation

Answer 49

Class II

Question 50

What happens to delta F508 as to why it cannot act functionally

WIDER READING - Cheng et al 1990

Answer 50

Fails to mature to its fully glycosylated form and is instead degraded

Question 51

Why can mutated CFTR fail to be transported correctly?

WIDER READING - Thomas et al 1992

Answer 51

Unestablished but if based on other proteins - mutations prevent it from adopting its correct conformational shape which results in the protein failing at quality control mechanisms and this causes protein degradation

Question 52

How can class II mutations be bypassed?

WIDER READING - Denning et al 1992

Answer 52

CFTR deltaF508 CFTR escapes the ER and is fully glycosylated in the golgi complex and delivered to the cell membrane when the temp is reduced from 37C to 23-30C this however is incompatible with life.

Function is around 30% of normal CFTR

Question 53

What are class III mutations in CF

WIDER READING - Welsh and smith 1993

Answer 53

Mutated proteins that have defective regulation.

Question 54

Give an example of a Class III mutation

WIDER READING - Anderson and welsh 1992

Answer 54

Mutation in the nucleotide-binding domain impairing their function such as ATP is less potent at stimulating activity of CFTR and complete activity too.

Question 55

What are class IV mutations in CF

WIDER READING - Welsh and smith 1993

Answer 55

Conduction issues.

Question 56

What happens in class IV mutations of the R117H

WIDER READING - Welsh and smith 1993

Answer 56

Conduction issues as Cl channel open for 1/3 of what wild types is.

Question 57

What part of the CFTR ensures that it favours anions (-) over cations (+) and how was this found out

WIDER READING - Sheppard and welsh 1999

Answer 57

Cysteine mutations (R352C) at the intracellular end of the pore result in a loss of anion attractiveness.

Question 58

What are the commonest pathogens to cause CF in children

WIDER READING - Davies 2002

Answer 58

Staph aureus and H.influenzae.

Chronic pseudomonal infection occurs in up to 85% of patients by adolescence

Question 59

What is the low-volume hypothesis for p. aeruginosa infections?

WIDER READING - Matsui et al 1998

Answer 59

In one, the low-volume hypothesis, sodium and water hyperabsorption depletes the volume of the ASL and mucus layer, within which cilia are unable to beat efficiently, and MCC is impaired. Allowing pathogens to settle in the mucus layer and cause infection and inflammation.

However - this defect alone would not explain the strong predisposition of the CF airway to infection with P. aeruginosa. Patients with other diseases in which MCC is impaired, such as primary ciliary dyskinesia or non-CF bronchiectasis have a much lower incidence of infection with this bacterium.

Question 60

What is the high-salt theory for p. aeruginosa infections?

WIDER READING - Smith et al 1996

Answer 60

Several salt-sensitive antibacterial defence proteins have been identified in the airway including β-defensins, lysozyme and lactoferrin.
A high-salt environment in the CF airway rendered these molecules non-functional

However - this defect alone would not explain the strong predisposition of the CF airway to infection with P. aeruginosa. Patients with other diseases in which MCC is impaired, such as primary ciliary dyskinesia or non-CF bronchiectasis have a much lower incidence of infection with this bacterium.

Question 61

Why is the low-volume theory favoured over the high-salt theory

WIDER READING - Knowles et al 1997

Answer 61

in vivo data have demonstrated that ASL electrolyte levels are not different in CF and healthy airways

Question 62

How may pH play a role in increased infection in those with CF

WIDER READING - Ballard et al 1999

Answer 62

ASL (airway surface liquid) pH. CFTR has recently been found to involve the transport of bicarbonate ions and thus may regulate cell surface pH. Certain mechanisms such as MCC and phagocyte function are pH-dependent and could be impaired in an acidic environment. Whether or not airway surface pH is abnormal in vivo remains to be confirmed.

Question 63

Why can P. aurginosa strains more susceptible to sticking on CF airways

WIDER READING - L. Saiman, A. Prince et al 1993

Answer 63

Receptors for pili and flagellae of the P.A bacteria bind to asialylated glycolipids and one of these, asialoGM1 (aGM1), is present in increased abundance on the surface of CF respiratory epithelial cells and many authors have reported increased adherence of P. aeruginosa to CF cells compared with those of wild-type (healthy, non-CF) origin

Question 64

Why is there low nitric oxide in CF patient lungs and how does this make them susceptible to disease

WIDER READING - Davies 2002

Answer 64

NO synthase expression is regulated by CFTR. NO has certain antibacterial properties and the low levels could be playing a part in the predisposition to infection.

Question 65

Why is glutathione important in CF for infection and infammation

Answer 65

the antioxidant, glutathione, is normally secreted via CFTR and is therefore deficient in the CF airway therefore increases inflammation

Question 66

Why are chronic P.A infections hard to treat

WIDER READING - Singh et al 2000

Answer 66

Initiation of biofilm formation is dependent on a process of “quorum sensing”.29 Bacteria produce molecules such as acyl homoserine lactones (AHLs) that diffuse freely in and out across the bacterial membrane. Due to this free diffusibility, the concentration within the organism reflects the concentration outside and enables the bacteria to “sense” other bacteria in the vicinity. Once a critical mass has been achieved, the quorum-sensing molecules induce the expression of the genes vital to biofilm production. In this state, microcolonies of bacteria are surrounded by a dense matrix, which protects against phagocytosis and prevents the penetration by antibiotic agents

Question 67

What role does CFTR have with aquaporin-3 channels in mammalian lung epithelial cells

WIDER READING - Schreiber et al 1999

Answer 67

CFTR, when stimulated by protein kinase A, activates a water permeability in respiratory epithelial cells. These results suggest coupling between Cl− transport as performed by the CFTR Cl−channel and water transport, thereby adjusting water permeability to electrolyte transport in the airway epithelium. In that sense, this mechanism would help to secure proper hydration of the surface fluid as well as mucociliary clearance

CFTR-dependent activation of AQP3 does not take place in airway cells carrying the CF defect or in cells overexpressing both AQP3 and mutant forms of CFTR. We may therefore speculate that regulation of AQP3 by CFTR is impaired in the respiratory tract of CF patients, which may contribute to the imbalance between secretion and absorption detected in CF airways

These findings were made using cell volume measurements of both CFTR positive and negative cells were taken with novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells.

Question 68

What is the relationship between ROMK2 and CFTR

WIDER READING - McNicholas et al 1997

Answer 68

Glebenclamide inhibits K currents into oocytes.

Using voltage patch clamp it was shown that - Glibenclamide inhibited K+ currents by 56% in oocytes expressing both ROMK2 and CFTR but only 11% in oocytes expressing ROMK2 alone.

Then looked at the NBF1 role by doing the same experiment with mutated NBF1 - In oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 46%. However, in oocytes coinjected with ROMK2 and a CFTR mutant truncated immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ currents by 12%.

This shows that CFTR has a role in ROMK2 channels and that it is due to NBF1 interaction. NBF1 motif is necessary for the CFTR-ROMK2 interaction that confers sulfonylurea sensitivity (glibenclamide is a sulfonylurea).

Question 69

What happened to sweat Cl conc in elexacaftor–tezacaftor–ivacaftor treatment

WIDER READING - Middleton et al 2019

Answer 69

he mean sweat chloride concentration in patients with a single Phe508del allele who received elexacaftor–tezacaftor–ivacaftor for 24 weeks decreased from 102 mmol per liter to 58 mmol per liter, just below the generally accepted diagnostic threshold for cystic fibrosis (≥60 mmol per liter)29; this finding reflects improved CFTR function.

Question 70

What is one side effect of elexacaftor–tezacaftor–ivacaftor treatment

WIDER READING - Middleton et al 2019

Answer 70

Slight rise in BP - likely due to salt preservation and improved nutrition

Question 71

What happened to exacerbations with elexacaftor–tezacaftor–ivacaftor treatment

WIDER READING - Middleton et al 2019

Answer 71

63% lower

Question 72

How does CF cause meconium ileus

WIDER READING - Sathe and Houwen 2017

Answer 72

Within the small intestine, CFTR is responsible for both Cl− and HCO3– excretion. It is the HCO3– that plays an integral role in chelating Ca2 + associated with the tight matrix of normally exocytosed mucins within the gut lumen to form normal, loose well-hydrated mucus [4]. Abnormal CFTR results in abnormal HCO3– secretion, thus decreasing luminal pH. This creates an acidic and dehydrated environment in which the tight matrix of exocytosed mucins are not disrupted appropriately resulting in thick, dehydrated mucus

Question 73

Give two types of drugs that can work to maintain cAMP signalling to CFTR

WIDER READING - Schultz et al 1999

Answer 73

phosphodiesterase inhibitors, phosphatase inhibitors - prevent the enzymes from breaking down cAMP.

However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.