Lecture 3 Flashcards
What is the structure of the influenza virion?
contains genetic sequence for many glycoproteins
o Matrix protein (M1)
o Haemagglutinin – binds to sialic acid residues – actn PKC & transient inhibition ENaC
o M2 – forms an acid activated, amantadine inhibited H+ channel, inserted into apical membrane host cell during infection
o Protons can move through the channel – changing the pH – impact the epithelial Na channel.
what did whole cell patch clamp and single channel current recordings show?
M2- inhibits function of ENac
Decreased open probability, also decrease channel number
• M2 decreases ENaC currents
• M2 decreases ENaC protein in membrane
how does M2 impact endocytosis?
M2 promotes endocytosis of epithelial Na channels from the apical membrane - decreases number of channels
How does M2 impact on reactive oxygen species?
m2 increase ROS
M2 reduces the number of channels and some of this is being mediated by an increase in ROS and stimulation of PKC.
What type of CFTR do you look for?
Band C CFTR - mature CFTR
What did an M2 knockout show?
DsiScram – inhibition – reduced amount of mature CFTR
DsiM2 (KO) – Amount of CFTR back up - currents back up to normal
Inhibition almost exclusively mediated by M2 protein – as in M2 ko theres no reduction in mature CFTR
Show that the M2 protein that’s made after infection is impacting on how much CFTR is available- reduces it and so reduces CFTR mediated currents, so reduces Cl secretion across the apical membrane
How does M2 impact on lysosomal degradation?
• In the presense of infection and M2, CFTR is targeted to lysosomes for degradation
how is the lysosomal degradation of CFTR tested?
infect with Udorn
Bafilomycin – prevents lysosomal acidification – inhibits lysosomal function
Lactacystin – prevents proteosomal degradation
Data suggests that the M2 protein is mediating movement of hydrogen ions, changing the pH, this targets mature CFTR to lysosomes to be degraded
How does M2 impact pH?
The changes in pH mediate inhibition of CFTR
amantadine inhibited H+ channel
Block H+ flux
Symptoms of PHA1
Salt wasting – lose Na in urine
Hypotension – water follows Na – ECF vol goes down
Hyperkalaemia – not reabsorbing as much Na at collecting duct principal cells – not secreting as much K – high plasma K
Metabolic acidosis – high H+
High renin & aldosterone
What are the features of the autosomal dominant form of PHA1?
Renal form
Problems localised kidney Mineralocorticoid receptor gene mutations
What are the features of the autosomal recessive form of PHA1?
Systemic form-
Multiple organs affected – esp airways
ENaC gene mutations - all subunits
Frequent lower respiratory tract illness
What is the impact of PHA1 on nasal surface liquid?
- Changes in the airway surface liquid layer in nasal epithelia – generate lots of liquid
- Inhibiting epithelial Na channel – liquid builds up – starts dripping out = cold
- PHA1 – runny nose all the time
- Excess conc of Na in the nasal discharge – defect in ENaC
Describe the impact of infection on the PCL and lung function. Explain the cellular mechanism that underpins the change in PCL?
- Infection may lead to an increase or decrease in the height of the PCL depending on factors – this shows increase.
- An increase in PCL reflects inhibition of ENaC is bigger than the inhibition of CFTR – balance favours chloride secretion
- Optimum height – 7.5um – this is higher – negative impact on function – excess volume that the cilia are trying to move by beating – physically cant manage to move bigger volume.
- Reduced mucous clearance – struggle to clear infection – at risk of secondary infection
