L5, CF: Gene to lung function

Question 1

Cystic Fibrosis: prevalence (carriers vs general), life expectancy, possible explanation for prevalence?

Answer 1

• 1 in 25 are carriers; most common genetic disorder
• Affects 1 in 2000 caucasians
• Life shortening condition; ~35 year expectancy, lower than 10 years if untreated
• Potentially advantageous in bacterial infections (e.g. Typhoid) -> Link to antagonistic pleiotropy, historical explanation for high prevalence

Question 2

Major clinical features of CF (x5):

Answer 2

• High salt concentration in sweat
• Pancreatic failure (exocrine pancreatic insufficiency; unable to wash out enzymes produced -> blockages)
• Gut unable to absorb nutrients (malnutrition)
• Lung is crippled with recurrent and persistent infections -> most deadly effect
• Infertility

Question 3

CFTR expression, nomenclature

Answer 3

• Expressed in epithelial cells, principally those of the pancreas, sweat glands, salivary glands, lung, intestine and reproductive tract
• Typically expressed on apical membrane of cell
• Named CFTR as it was originally thought to be a regulator of chloride permeability and not an ion channel

Question 4

Structure of CFTR:

Answer 4

• TM domain 1 and 2
• Nucleotide binding domains 1 and 2
• Regulatory domain (phosph. site)
• TRL: Regulation of other transporters and proteins; signal transduction role
• Diagram in notes/FCs

Question 5

Evidence that CFTR functions as a channel: (5 points regarding electrophysiology, expression experiments etc)

Answer 5

• Expression of CFTR in a variety of heterelogous expression systems produces current similar to that measured in epithelial cells
• Activation depends on both cAMP-dependent protein kinase A and ATP binding at NBDs
• Channel with these properties is missing in CF sufferers
• Purified protein incorporated into artificial bilayers give chloride currents
• Mutation of the channel itself have been shown to alter channel activity properties
• Co-expression of two different mutants of CFTR channels with different functional characteristics - no hybrid channels produced (therefore MONOMERIC)

Question 6

Classes of CFTR defect: Detail and background

Answer 6

• > 1000 mutants known
• 4 main classes…
• Class I: Defective protein synthesis (truncations caused by frameshifts)
• Class II: Defective trafficking of proteins -> loss of CFTR expression in membrane
• Class III: Defective regulation of channel
• Class IV: Altered ion permeation
• Class V: Insufficient protein
• Class VI: Accelerated turnover

Question 7

+ List the 5 classes of CFTR mutant by affect

Answer 7

• Protein production
• Protein processing (trafficking)
• Gating
• Conduction
• Insufficient protein
• Accelerated turnover

Question 8

2 examples from different CFTR mutant classes (Classes II and VI):

Answer 8

• Most common (>65%) is F508del (Class II); drops phenylalanine, leading to a minor misfolding of protein that, in golgi, gets stuck and is not released as a vesicle for transport to PM
• Q1412X: C-terminal truncation (Class VI)

Question 9

Account for the varying severities between CFTR patients:

Answer 9

• Mutations leading to total LOF -> severe form
• Mutations resulting in reduced Cl- current -> milder form (e.g. reduced single channel conductance, reduced open probability or reduced levels of protein expression)

Question 10

How does CFTR interact with other ion channels? -> 2 examples with structural detail

Answer 10

• TRL domain of CFTR thought to interact
• ENaC: Na+/H+ permeable -> target for ability to retain salt; complex ligand gating, amiloride sensitive; downregulation of sodium conductance
• CaCC: Calcium-activated Cl- channels , 8TMS; ubiquitous in animal cell PM e.g. in frog oocyte expression; upregulation of chloride uptake

Question 11

Pathophysiology of CF in the lung:

Answer 11

• Unremitting and inappropriate absorption of the ASL -> collapse of periciliary layer and mucus layer
• Hyperviscous mucus layer in lungs -> static, accumulation of bacteria on biofilm, bacterial DNA adds to viscosity
• Eventual infection

Question 12

CF therapies:

Answer 12

• Hydrating lung by inhaling hypertonic saline (temporarily relieving symptoms)
• Administer drugs to affect transporters -> redirection of transport to secretory direction

Question 13

Ivacaftor:

Answer 13

• Increases open probability of CFTR channel
• Effective for ~ 5% of CF patients
• More effective when mixed with drugs that help correct the malformed CFTR trapped in golgi -> Aiding transport to PM

Question 14

How do mutations in CFTR in sweat glands cause symptoms of CF?

Answer 14

• Na/K ATPase in the basolateral membrane creates and e/c gradient for Na+ uptake across apical membrane
• Cl- follows passively via CFTR to maintain neutrality
• Duct epithelium is impermeable (tight junctions) to water -> leaves via duct to skin
• In CF, L- uptake is inhibited, so Na+ uptake cause membrane depolarisation, reducing driving force for Na+ uptake from duct -> reduced NaCl absorption
• See notes for diagram

Question 15

CFTR mutations in the lung: How is ASL normally maintained vs in CF patients?

Answer 15

• Epithelial duct permeable to water -> follows balance of solute
• Basal conditions: water absorbed from AS L (basolaterally expressed Na/K ATPase sets up e/c gradient, ENaC -> Na+ influx, CFTR -> Cl- influx
• ASL too thin: upregulation of CFTR, subsequent inhibition of ENaC; more Cl- efflux, lack of Na+ influx -> water and Na+ pass through tight junction
• CF sufferers: decreased capacity for Cl- efflux and constant, unregulated Na+ uptake -> unremitting and inappropriate absorption of the ASL