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A BMS346 Epithelial Physiology > Influenza II and PHA > Flashcards

Influenza II and PHA Flashcards

1
Q

What genetic sequences does the influenza virus contain?

A

For many glycoproteins:

  • Matrix protein (M1)
  • Matrix protein (M2)
  • Haemagglutinin
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2
Q

What does the M2 of the influenza virus do?

What does this impact?

A

Forms an activated, amatadine inhibited proton (H) channel that is INSERTED into the APICAL membrane of the HOST cell during infection

Protons move through the channel and impact on the function of the ENaC channel

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3
Q

What impact does M2 over expression have on ENaC currents?

How were these currents measured?

A

Reduced ENaC currents compared to the control (non-transfected, GFP expressing cells)

Measured using whole cell current measured with the patch clamp

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4
Q

How can M2 over expression reduce the currents of ENaC?

A
  • Reduces Po

- Channel number

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5
Q

What is the evidence that M2 decreases ENaC protein in the membrane?

A

Looking at the alpha and beta subunits in a Western Blot:
- In the absence of M2

  • In the presence of M2
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6
Q

What occurs to the alpha and beta subunits in the Western Blot analysis in the presence of M2?

What does this show?

A
  • Density of both bands are REDUCED
  • Larger reduction in the beta band

Shows:
- Reduction in the N number

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7
Q

What aspect of influenza does the over expression of the M2 protein mimic?

A

Mimicks one part of the infection, rather than looking at the direct infection

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8
Q

What could the reduction in the N number of ENaC channels in the membrane be a result of?

A
  • Channels not getting to the membrane

OR

  • Channels being removed from the membrane (endocytosis)
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9
Q

What happens to the current in the presence of ENaC and M2 compared to M2 alone?

A

Huge reduction in current recorded (almost no current) in ENaC and M2

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10
Q

How can you tell if M2 promoted the removal (endocytosis) of ENaC from the membrane?

A

Exploit a Liddle’s mutant version of ENaC (Liddle’s mutant is RESISTANT to endocytosis):

If over express the mutant and endocytosis has NOTHING to do with the response –>

Would see the SAME reduction in the current as in the WT ENaC channel

If over express the mutant and endocytosis is promoted by the M2 protein –>

Won’t be as big of a DROP in the current (Liddle’s mutant prevents endocytosis –> reduce inhibition by M2)

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11
Q

Does M2 promote the endocytosis of the ENaC channel?

How is this seen?

A

Yes - a significant amount of the reduction in the ENaC current in the presence of M2 is due to promoting endocytosis

Reduction in the current but not the extent of the WT

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12
Q

What molecules may be involved in the promotion of endocytosis of the ENaC channel from the membrane n the presence of M2?

A

ROS and PKC

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13
Q

How is it seen that ROS may be involved in the promotion of endocytosis of the ENaC channel from the membrane n the presence of M2?

A

Seen using RFP-M2 and GFP (that shows the presence of ROS):

  • Co localisation data shows that where M2 is expressed is the same position as where ROS is made (glows orange)
  • ROS is not made in RFP only cells (No M2)

However, NOT sufficient to say that M2 CAUSES ROS increase (cause doesn’t mean consequence - may be incidental)

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14
Q

What is seen in GFP-M2 cells that are exposed to GSH?

Why?

What does this show?

A
  • INCREASE the ENaC current (reversal of the M2 dependant reduction of the ENaC current)
  • Due to the reversal of the increase of ROS increase as GSH is an antioxidant
  • Reducing inhibition of ENaC

Shows that the over expression of M2 increases ROS production that have a role in the inhibition of ENaC

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15
Q

How is it seen that PKC may be involved in the promotion of endocytosis of the ENaC channel from the membrane n the presence of M2?

A

Using inhibitors of PKC (Go6796 and inhibt):

  • If PKC is involved in the M2 dependant inhibition of ENaC
  • -> inhibition of PKC will reverse the inhibition of ENaC (this is the case)
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16
Q

So, overall what does the overexpression of M2 cause and how?

A
  • Decrease in Po of ENaC
  • Decrease in the N of ENaC channels in the membrane (by endocytosis)
  • Some of which is meditated by ROS and PKC
  • ROS increase
  • PKC stimulation
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17
Q

How do ROS and PKC interact?

A

Not yet sure, they COULD be in the same pathway

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18
Q

Why is the impact on the ASL by influenza complex?

A

Different studies of influenza show alternative ideas:

  • Some show the hight of the ASL to increase
  • Some show the height of the ASL to decrease
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19
Q

What are the differences that may explain why different studies show the ASL to increase/decrease?

A

In different studies:

Different CELL SOURCE

  • STRAINS of mice
  • Difference in the genetics of humans and how we respond to influenza (some have more serious symptoms than others)

Influenza VIRUS PROPERTIES

  • Different influenza viruses have different properties

Different LAB CONDITIONS (experimental conditions)

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20
Q

As well as ENaC, which other channel function is inhibited with infection by influenza?

A

CFTR

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21
Q

How is CFTR activated with Forskolin?

A

Forskolin activates cAMP that activates CFTR

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22
Q

What is the response of CFTR to Forskolin a measure of?

A

The function of the CFTR channel and Cl secretion

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23
Q

What is the difference in the SCC when treat the cells with Forks in infected/non-infected cells?

What does this show?

What is this response with GlyH and Amiloride?

A

Non infected cells:
- Large increase in current when exposed to Forks

Infected cells:

  • No increase in current with Forks
  • Shows an inhibition of the CFTR channel (unable to respond to Forks)

Reduces amiloride and GlyH sensitive SCC in the infected cells - shows inactivation of ENaC and CFTR prior to treatment

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24
Q

What does GlyH do?

A

Inhibit CFTR

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25
Q

What happens when run a western blot for CFTR? Why?

A

Get 2 bands:

Band B

  • Low molecular weight
  • Immature CFTR
  • Less glycosylation

Band C

  • High molecular weight
  • Post-golgi, traffics to the membrane
  • Mature CFTR/functional CFTR
  • More glycosylation
26
Q

When determining the function of CFTR with Western blot, what you look at?

What does the over expression of M2 cause?

A

Look at the density of band C/amount of band C (functional/mature CFTR)

Over expression of M2 causes a reduction in band C (reduces mature CFTR levels) in both the whole cell and at the MEMBRANE

27
Q

What is the use of siRNA in looking at the function of CFTR?

A

siRNA –> directed to mRNA, so unable to translate the mRNA into protein:

1) DsiScram (Scrambled - control)
2) DsiM2

28
Q

What is the use of DsiScram?

A

Scrambled siRNA

Non-targeting

Used as a CONTROL (to ensure it isn’t the experimental approach that causes the change in response

29
Q

What is the use of DsiM2?

A

Targeted to M2 –> M2 functional knockdown

30
Q

What is the affect on CFTR band C with DsiScram and DsiM2 in the uninfected cells?

Why?

What is the affect of CFTR current?

A

There is no significant difference in CFTR band C between DsiScram and DsiM2

Because the M2 virus comes from the virus

Affect on current:
- CFTR currents are reduces

31
Q

What is the affect on CFTR band C with DsiScram and DsiM2 in the infected cells?

Why?

What is the affect of CFTR current?

A

In DsiScram - the amount of CFTR band C is reduced (not targeted to M2, M2 is functional)

In DsiM2 - CFTR band C levels return back to normal (M2 is inhibited)

Affect on current:
- CFTR currents are inhibited with DsiScram (M2 is active)

  • CFTR currents are restored with DsiM2 (M2 is inactivated)
32
Q

What is the inhibition of the CFTR channel in influenza mediated by?

How know this?

A

Almost entirely mediated by M2

If KD M2 protein (still have all the other infection impacts) –> there is NO REDUCTION in the amount of the CFTR protein (no longer any affects as a result of influenza)

This recovery of CFTR is also shown to be functional

33
Q

If KD M2 - the CFTR level returns to normal, but it the protein functional?

How can this be seen?

A

YES - protein is functional

Currents through the CFTR channel are restored with the levels of the CFTR band C return to normal with M2 KD

34
Q

How does M2 impact CFTR after infection?

A

Impacts on how much mature CFTR is available:

  • Reduces the CFTR
  • Reduces the CFTR mediated currents (Cl secretion across the apical membrane)

–> Impacts on the heigh of the ASL (reducing it)

35
Q

What is the M2 protein?

A

A AMANTADINE INHIBITED proton (H) transporter that are made after infection and reside in the apical membrane

36
Q

Are all M2 proton channels made as a consequence of infection amantadine sensitive?

Why?

A

No (some aman aren’t sensitive)

Due to differences in the coding sequence and AA sequences in the channel

37
Q

What is an example of a virus that causes the host cells to make an aman sensitive M2 protein?

A

The Udorn virus

38
Q

What happens in the cells if add aman after the cells are infected with Udorn?

A

Udorn - makes M2 protein (protien channels) that are aman sensitive

  • Therefore, aman blocks the M2 protein from transporting H ions (no H flux)
  • -> ion channels aren’t functional
39
Q

What is the effect of aman in uninfected cells?

Why?

A

No effect - control experiment

Uninfected cells - no M2 (cannot be blocked)

40
Q

What happens to cells when infected with Udorn? (no aman)

A

Reduction in the CFTR band C in the membrane

–> Inhibition of the CFTR channel

41
Q

What happens to cells when infected with Udorn? (WITH aman)

What does this show?

A
  • Aman blocks M2 function
  • Amount of CFTR band C in the membrane increases (functional channel)
  • With increasing concentration of aman –> increasing concentration of CFTR band C in the membrane

Shows:
- That proton movement and pH changes must be influencing what is happening to the fully mature version of CFTR

42
Q

What is an example of a virus that leads to M2 protein that is NOT aman sensitive?

A

The PR8 virus

43
Q

What happens in the cells infected with the PR8 virus in the absence and presence of aman?

What does this show?

A

Presence of aman:
- Redcution in the amount of CFTR band C (mature CFTR)

Absence of aman:

  • M2 functions NORMALLY
  • H transport still occurs through the channel
  • NO RECOVERY in mature CFTR levels

Shows:
- H ion transport and pH changes are playing a role in what is happening to the mature CFTR channel upon infection

44
Q

What evidence suggests that the mature CFTR channel may be targeted to lysosomes for degradation in the presence of M2?

What does this also suggest?

A

Look at the level of CFTR band C in the presence of:

1) BAFILOMYCIN (prevents lysosomal acidification)
2) LACTACYSIN (prevents proteasomal degradation)

No effect in uninfected cells with vehicle, baf or lac

In the infected cells:

  • Vehicle reduces CFTR band C
  • Baf reverses this reduction, Lac has no affect

ALSO SUGGESTS:
- M2 protein is mediating the movement of H ions, changing the pH

45
Q

What happens if prevent lysosomal acidification?

A

Prevents lysosomal function

46
Q

What is suggested about pH?

Why?

A

pH is somehow targeting the mature CFTR to lysosomes, where it is degraded

As when lysosomal acidification is prevented, CFTR band C levels recover

47
Q

What are the different types of mutations in ENaC?

Examples?

A

GAIN of function:

  • Liddle’s syndrome
  • ->HYPERtension

LOSS of function:
- PHA –> HYPOtension

48
Q

What is the impact of Liddle’s syndrome on ENaC?

How?

A

INCREASE in ENaC (channels are resistant to endocytosis –> stay in the membrane longer than they should)

Due to changing the interaction between ENaC and Nedd4 (a ubiquitin ligase family)

  • Channels unable to be ubiquitinated
  • Unable to be retrieved from the membrane
49
Q

What is the impact of PHA on ENaC?

A

ENaC inactivation

50
Q

What are the 5 symptoms of PHA type 1?

A

1) Salt wasting
2) HYPOtension
3) HYPERkalemia
4) Metabolic acidosis
5) High renin and aldosterone

51
Q

Why does PHA type1 cause salt wasting?

A
  • ENaC is inactivated
  • Na cannot be reabsorbed
  • Na is lost in the urine
52
Q

Why does PHA type1 cause HYPOtension?

A
  • Water follows Na
  • Na lost in the urine –> water lost in the urine
  • Extracellular volume compresses and the volume goes down
53
Q

Why does PHA type1 cause HYPERkalemia?

A
  • Not as much Na reabsorption across the collecting duct principle cells
  • Not secreting as much K –> K accumulates
54
Q

Why does PHA type1 cause metabolic acidosis?

A
  • Na reabsorption across the collecting duct normally drives H ion SECRETION into the urine (+ve charge)
  • Not as much Na absorption –> not as much H secretion
  • High H conc (acidosis)
55
Q

Why does PHA type1 cause high renin and aldosterone levels?

A
  • Body knows it is losing Na
  • Compensate by the upregulation of renin and aldosterone (mechanism to try and retain Na)
  • Depending on the mutation - this renin aldosterone mechanism DOESN’T work
56
Q

What are the 2 types of PHA1?

A

Autosomal dominant (RENAL form)

Autosomal recessive (SYSTEMIC form)

57
Q

Describe the autosomal dominant form of PHA1 (problems, cause)

A

RENAL form:
- Problems localised to the KIDNEY

  • Associated with mutations in the mineralocorticoid receptor gene (a target for aldosterone)
58
Q

Describe the autosomal recessive form of PHA1 (problems, cause)

A

SYSTEMIC form:
- Multiple organs affects (eg. the airway)

  • Caused by ENaC gene mutations (mutations in all ENaC subunits seen)
  • Frequent lower respiratory tract illness and constant sore/runny nose (due to disruption to the cilia)
  • Increased ‘wet rate’, nasal passage are quite closed and full of liquid
  • Concentration of Na is in excess in the nasal discharge (less Na reabsorption)
59
Q

Why do patients with PHA1 have a constant runny nose when have cold/flu?

A

Due to changes in the nasal epithelium:

  • Inhibition of ENaC MUCH MORE than CFTR

–> Reduces Na/water reabsorption –> generate lots of liquid

60
Q

What happens to the Vte in systemic patients?

What happens to the % inhibition with amiloride?

What does this show?

A

It is reduced (closer to 0mV)

% inhibition is also reduced

Shows ENaC function to be reduced

61
Q

What is the difference in % inhibition with amiloride between normal/renal patients and systemic patients?

A

Normal/renal:
60%

Systemc:
5%

62
Q

How may the function of ENaC be reduced?

A

Number of channels

Open probability (doesn’t function efficiently at the membrane)

A BMS346 Epithelial Physiology (13 decks)

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