L3 - Influenza II and PHA

Question 1

Influenza virion contains the …

Answer 1

Genetic sequence for many glycoproteins

Question 2

Give some examples of the glycoproteins encoded by Influenza

Answer 2

M1
Haemagglutinin
M2

Question 3

What is M2

Answer 3

An acid activated amantadine sensitive H channel which is inseerted into the apical membrane early in infection

Question 4

What is the question RE M2

Answer 4

Is it involved in the long term regulation of ENaC

Question 5

OE M2 - what is the effect on whole cell current

What was the control

Answer 5

CONTROL - OE GFP

OE M2 seen to inhibit ENaC show by a decrease in currents

No effect on the GFP OE (contro)

Question 6

OE M2 - effect on single channel recordings

Answer 6

OE M2 Decreases the Po of the channel

hint in the data that this may be due to changes in N number

Question 7

OE M2 Western blot

Answer 7

Density of band reduced for both a-ENaC and b-ENaC

Question 8

What is the Liddles ENaC resistant to

Answer 8

Endocytosis

Question 9

Hypothesis for how OE M2 was decreasing the number of channels in the membrane

Answer 9

Could be by removal from the membrane by endocytosis

Question 10

How was it tested to see if M2 was causing an increase in endocytosis of ENaC

Answer 10

Using Liddles - ENaC (RESITANT TO ENDOCYTOSIS)

Leads to a reduction but not by as much!
Suggests that M2 is causing an increase in endocytosis

Question 11

Describe the experiments and results to see if ROS were involved

Answer 11

OE RFP - ROS-GFP
OE M2 shows increased levels of ROS (not all since transfection not 100%)
Colocalisation shows M2 expressing cells also upregulate ROS

Question 12

Describe the experiments looking at PK-C

Answer 12

ENaC currents - OE M2-GFP

OE M2 inhibits ENaC currents

Question 13

What is glutathione (GSH)

Answer 13

Anti-oxidant

Question 14

Why was GSH used

What were the results

Sum this up relate to M2

Answer 14

Bring down levels of ROS - if see recovery then shows ROS involved

Saw recovery

OE of M2 protein increases ROS levels and this is playing a role in the inhibition of ENaC

Question 15

What is Go6796

Answer 15

PK-C inhibitor

Question 16

What was seen when go6796 was added

Answer 16

If OE M2 then if PKC is involved then inhibiting PKC should relieve this inhibition and recovery was seen indicating a role for PK-C

Question 17

Sum up the role of M2

Answer 17

OE M2 is working by reducing the Po and N of ENaC and some of this is being mediated by an increase in ROS and stimulation of PK-C

This may be in the same or different patwhays.