Cystic Fibrosis Flashcards
What is Cystic Fibrosis (CF)?
= most common genetic disorder
(1/25 are carriers)
(may be linked to typhoid prevention)
= life-shortening condition with average life expectancy of approx. 35 years
(untreated - life expectancy = <10 years)
Major Clinical Features:
= High salt concentration in sweat (excess NaCl)
= Failure of the pancreas (exocrine pancreatic insufficiency)
= Gut unable to absorb nutrients (malnutrition)
= Lung crippled with recurrent and persistent infections (killer)
= Infertility
What is the CFTR?
= cystic fibrosis transmembrane conductance regulator
= CF patients have impaired Cl- absorption
= gene identified: 27 exons encoding 1480 a.a
(mapped to chromosome 7 - 7q31)
= expressed in epithelial cells principally those of the pancreas, sweat glands, lungs, intestine and reproductive tract
= transmembrane spanning domains - homology to ABC transporter family
What is the structure of CFTR?
= transmembrane spanning protein
5 main domains
= R-domain (regulatory domain) located on cytoplasmic side = contains sites for phosphorylation by protein kinases
= NBD1 (nucleotide-binding domain 1) = cytoplasmic site = binds ATP = site of the common ΔF508 mutation
= TMD1 (transmembrane domain 1) = spans cell membrane
= NBD2 (nucleotide-binding domain 2) = cytoplasmic side = binds ATP
= TMD2 (transmembrane domain 2) = spans cell membrane
TMDs = form pore for Cl- ions to pass
R-domain = regulates opening and closing of pore
(phosphorylated = conformational change = opens pore)
What is the function of CFTR? (+Evidence)
Evidence that CFTR functions as a channel:
- Expression of CFTR in many heterologous expression systems produces currents that resemble epithelial cells (more selective for Cl- over Na+, voltage independent)
- Activation dependent on both cAMP-dependent protein kinase A (PKA) and ATP binding at NBDs
- Channel with these properties is missing in CF sufferes
(found using patch clamp / electrophysiology) - Purified protein incorporated in to artificial bilayers give chloride currents
(does NOT require ‘accessory’ subunits for basic channel activity) - Mutations in the channel itself shown to alter channel activity properties
(e.g. mutation in NBD1 = decreased frequency of opening)
(e.g. mutation in NBD2 = prolonged duration of opening events) - Co-expression of two different mutants of CFTR channels with different functional characteristics = no hybrid channels produced
= therefore a MONOMER
What are some examples of mutations of the CFTR?
= >1000 known mutations, BUT 4 main classes of defects:
Class I
= defective protein synthesis
= truncations caused by frame-shifts as a result of nucleotide insertions, deletions or mutations
Class II
= defective trafficking of protein resulting in loss of CFTR expression in membrane
= most common is ΔF508 (>65%)
Class III
= defective regulation of the channel
= e.g. G551D , lower affinity for ATP
Class IV
= altered ion permeation
= e.g. R347P
How do mutations affect the severity of CF symptoms?
Mutations leading to total loss of functional activity
= e.g. protein incorrectly processed + missing from plasma membrane
OR present but completely inactive
= SEVERE form of the disease
Mutations which result in reduced Cl- current
= e.g. reduced single channel conductance, reduced open probability
OR reduced levels of protein expression
= associated with MILDER form of the disease
Why is CFTR more than just a channel?
= It regulates other ion channels + more
e.g. ENaC
= Na+/H+ permeable
= 4 subunits (2 alpha, 1 beta, 1 gamma) = each with two TMS
= major role in regulating Na+ content of blood and epithelia fluids
= complex ligand-gating
= amiloride sensitive
= downregulated by CFTR
e.g. CaCC
= Ca2+ activated Cl- channels (anoctamins)
= 8 TMS
= ubiquitous in animal cell PM
= upregulated by CFTR (increases permeability of Cl- into cell membranes
EXTRA READING (other functions)
= regulation of cell proliferation and apoptosis (implicated in some cancers)
= interactions with other proteins = cellular transport and signalling pathways
= modulation of the immune response = production of cytokines / activation of immune cells
How do mutations in CFTR cause CF symptoms? (pathophysiology) in the sweat glands
(occurs in the sweat glands)
- Na/K ATPase
= in basolateral membrane
= creates electrochemical gradient for Na+ uptake across apical membrane - Cl- follows passively (duct lumen negativity) via CFTR (apical membrane)
= maintains electoneutral NaCl uptake - Duct epithelium is impermeable to water
= leaves via duct to skin - In CF
= Cl- uptake from duct is inhibited
= Na+ uptake causes membrane depolarisation
= which reduces driving force for Na+ uptake from duct
= hence reduced NaCl absorption
How do mutations in CFTR cause CF in the Lungs?
Fluid layer in lung airway
= results from fine balance between fluid uptake and secretion
= main morbidity / mortality in CF is chronic lung infections / deteriorating lung function
= associated with hyperviscosity of the lung fluid
(unable to remove bacteria = infection)
Mechanical clearance is primary defence mechanism for airways
= funnelling of ASL (airway surface liquid) up converging airway surface
= water passes through tight junctions
= ions pass through channels
(CFTR = Cl-, ENaC = Na+, Na/K+ pump also)
How is adequate ASL (airway surface liquid) maintained?
= links to how mutations in CFTR cause CF symptoms in lungs
- Na+ influx and Cl- efflux across apical membrane
= balanced to maintain ASL volume
= lung has capacity to secrete or absorb water - Basal conditions
= water needs to be absorbed due to converging surfaces
= greater Na+ influx drives paracellular Cl- and H20 absorbance - If ASL becomes too thin
= upregulation of CFTR
= inhibition of ENaC
= maintains sufficient ASL (self-regulation) - In CF
= decreased capacity for Cl- efflux
= constant Na+ uptake - Results in unremitting and inappropriate absorption of the ASL
= collapse of periciliary layer
= mucus layer sticks to the epithelial layer
= results in obstruction of airways, inflammation and chronic infections
Chronic persistent infections
= mucus layer forms a biofilm ideal for growth of bacteria
= increased thickness of mucus layer reduces diffusion of antibacterial agents released by lung epithelia
= neutrophils can’t diffuse freely to capture and kill bacteria
What is the evidence for ASL volume depletion in CF?
- Cultures of lung epithelial cells (at air liquid interface)
= maintain adequate ASL volume for long periods of time (through Na+/Cl- regulation)
Equivalent CF cultures are locked into unregulated Na+ absorption mode
= with little capacity for Cl- secretion
- Mouse Model
= express little CFTR in lungs
= engineered mice to have enhanced Na+ absorption exhibit depleted ASL and mucus obstruction
= 50% of mutant mice die within 30 days and present CF lung disease symptoms
How has the ASL volume depletion hypothesis been modified?
Limitation of a simple ASL volume depletion model:
= predicts lungs of babies with CF will deteriorate rapidly soon after birth
= BUT data shows CF symptoms take many years to present
Current hypothesis:
= there is sufficient Cl- transport in most conditions
= due to other Cl- channel activity (esp. CaCC)
= BUT during stress (e.g. infection) CF lung has increased vulnerability = causing collapse of ASL and CF symptoms
Normal lung
= rehydration of lung via CaCC and CFTR
= when ASL becomes too thin CFTR downregulates ENaC + reduced Na+ influx
= therby increasing ASL volume (rehydration of lung surface)
= therby countering CF symptoms
CF lung
= reduced Cl- efflux
= under most conditions there is sufficient Cl- permeability (via CaCC) to maintain ASL volume
= BUT under viral stress (e.g. infection increasing viscosity of ASL)
= leads to upregulation of Na+ influx in absence of CFTR
(normal lung = CFTR is present to boost Cl- efflux and downregulate Na+ influx via ENaC)
What do therapies target?
= directed at airway surface rehydration
- Airway surfaces are relatively permeable to water
= so to rehydrate = add salt to the CF airways
e.g Inhale hypertonic saline (HS)
= 7% salt solution
= twice daily
= shown to reduce infections and improve lung functions
- Administer drugs to affect transporters which result in redirection of transport to secretory direction (some in clinical trials)
= e.g. Moli 1901 = formulation of duramycin
(antibiotic thought to form anion channels in membranes / activate anion channels)
= e.g. SPI-8811 = chloride channel opener (CLC-2)
= e.g. PS552 = amiloride analogue which acts to block ENaC
= e.g. INS37217 = metabolically stable UTP derivative which activates P2YP2 receptor (which inhibit ENaC and activates CaCC)
What is an example of a licensed therapeutic agent that act directly on the CFTR?
= Ivacaftor (a CFTR modulator)
= increases open probability of CFTR channel
= effective for approx. 5% of CF patients that express channel but has defective gating
(e.g. G551D, G551S, G178R, S549N)
= has recently been formulated with other drugs (e.g. lumacaftor) that help to correct malformed CFTR trapped in golgi (ΔF508) + aid transport to the PM
(has wider spectrum for effectiveness)
EXTRA READING (other treatment options)
= gene therapy (introduce healthy copy of CFTR)
= anti-inflammatories (block certain cytokines)
= mucus clearing agents (HS)
= anti-infectives (can target specific bacteria that commonly infect CF patients)
