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BMS346 - Epithelial Physiology > L8 - K Channels and Epithelia > Flashcards

L8 - K Channels and Epithelia Flashcards

1
Q

What is the effect of K channels opening in the membrane

What is their function

A

K moves out of the cell and the Vm is driven towards EK

Maintain and negative Vm

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2
Q

What else do K channels have a role in

A

Cell volume

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3
Q

Describe the voltage gated Kv channel family

A

4 subunits - 1 channel
Pore region
6 TMD per subunit, number 4 is the voltage sensor

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4
Q

Describe the inwardly rectifying K channel family

A

4 subunits of 2 TMD must come together to from a functional pore
Hyperpolarise the Vm when open
2 TMD and a pore region

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5
Q

Describe the two pore domain K channel family

A

4 TMDs and 2 pore regions per subunit
2 subunits form a channel
Consitiutive activation

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6
Q

What do the 2PD K channels have a role in

A

Important in setting the resting Vm

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7
Q

Give some examples of Kv channels found in epithelial

A

KCNQ1 - regulated by the Ex subunit

KCNA10 - found in the proximal tubule

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8
Q

Give some examples of Kv channels that are Ca activated

A

Sk4 and BK

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9
Q

What other classification is there for the SK4 and BK channels

What does this mean?

A

Classified based on conductance
Low - mid - high

This is the number if ions that flow through the pore per unit time

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10
Q

Example of Kir channel and its location

A

Kir 1.1 (ROMK) in the kidney

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11
Q

Examples of two pore domain K channels

A

TWIK-1

TASK-2

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12
Q

Describe the apical membrane of the upper airway cell - including K channels

A 
APICAL 
Ca activated chloride channel (secretion of Cl)
ENaC
CFTR 
BK K channel (K secretion)
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13
Q

Describe the basolateral membrane of the upper airway cell - INCLUDING K CHANNELS

A 
BASOLATERAL 
SK4 - K channel - K out 
Na/K ATPase
NKCC1 (Na, K, 2Cl cotransporter (all IN)
KCNQ1 (KvLQT1) - E3
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14
Q

What is CaCC activated by

A

Ca

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15
Q

What is SK4 activated by

A

Ca

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16
Q

What is CFTR activated by

A

cAMP

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17
Q

What are BK channels activated by

A

Ca

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18
Q

What is KVLQT1

A

KCNQ1

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19
Q

What is the beta subunit E or Q

A

E

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20
Q

What is KCNQ1 activated by

A

cAMP

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21
Q

How many pathways are their to trigger Cl secretion in upper airway cells

A

2

One cAMP and one Ca

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22
Q

Describe the primary pathway for Cl secretion - involving CFTR

A

Increase in cAMP
Opening of KCNQ1 - K secretion
Hyperpolarisation
Driving force for Cl secretion - through CFTR - also activated by cAMP

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23
Q

Describe how the short circuit current due to K movement can be measured

A

Permeablise the basolateral membrane
Apply gluconate (low chloride) - driving force for Cl secretion
Can then measure the Isc mediated by K movement

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24
Q

What technique would be used to measure the short circuit current

A

Ussing Chamber

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25
Q

Describe the secondary pathway of Cl secretion

A

Increase in IC Ca
SK4 (mid conductance) and BK (high conductance) K channels are activated
Hyperpolarisation of the Vm
Also activated Ca activated Cl channels
THIS IS THE SECONDARY PATHWAY FOR CL SECRETION ACROSS THE APICAL MEMBRANE

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26
Q

If CFTR was the only Cl channel in apical membrane … what would we predict in CF

Why is this?

A

Predict when mutant that mucociliary clearance would stop

Have CaCC channels which can be upregulated to compensate

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27
Q

Why does the mouse not make a good CF model

A

CaCC are able to take over from the mutant CFTR sufficiently so that Cl secretion and mucociliary clearance is not significantly impaired

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28
Q

Describe how the short circuit current due to K movement can be measured

A

Permeablise the basolateral membrane
Apply gluconate (low chloride) - driving force for Cl secretion
Can then measure the Isc mediated by K movement

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29
Q

Describe what is meant by “looking at ATP activated currents”

A

Actually measuring CA ACTIVATED K SECRETION

Since ATP activates purine receptors P2Y and P2X –> increase in Ca

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30
Q

What does it mean if there are large ATP activated currents

A

Then there is a big function of K channels

31
Q

What is paxilline

A

Inhibitor of BK channels at the apical membrane

32
Q

What is seen to Isc upon the addition of paxilline

Why

A

Decrease in currents

Decrease Ca activated K secretion since since BK channels on the apical membrane

33
Q

What sort of experiment was used to verify that BK channels are important for ATP activated ISC

A

Use a knockdown experiement of the BK channels

34
Q

What were the three experimental conditios of the BK knockdown expt

A

NI - not infected
NT- using scrambeled shRNA
KD - using targetting shRNA

35
Q

What were the results of the BK KD

A

No difference between NI and NT - expected

In knockdown see significantly decreased currrents

36
Q

Describe the mechanism for looking at the ciliary beat frequency

A

Plate onto inserts
Remove the fluid from the apical membrane
Forms and air liquid interface
Lack of liquid causes cells TO PRODUCE THEIR OWN ASL
Measure ciliary beat frequency
Add PBs to increase height of the layer and measure again

37
Q

In the BK - CBF study what was the test and control conditions - how did this address the question

A

Presence or absence of paxilline (blocks the BK channels)

If the BK channels are important we would expect the ASL not to increase to optiumum - cilia bend over and CBF decreases

38
Q

What was ‘part 2’ of the BK - CBF expt

Why was this done?

A

Non targeted and a knockdown

Dfferent way of doing the same experiement

39
Q

Describe the BK_CBF data for when paxilline date

A

Control CBF of 10Hz
Then test CBF see decline (since BK blocked, Cl secretion inhibited)
Then add PBS - CBF starts to increase

40
Q

Describe the BK-CBF data for KD

A

Same results as before

41
Q

Impact BK channel function ….

A

Impact on Cl secretion

Impact on the height of the ASL

42
Q

Describe the impact of cigarette smoke on CFTR and BK channels

A

Take HBE cells from smokers and non smokers

Culture until fully differentiated - air liquid interface

43
Q

How was CFTR activity measured

A

Using the Cl gdt - whole cell currents

44
Q

How was BK activity measured

A

Permeabilise the basolateral membrane
Use gluconate solution
K gradient

45
Q

Effect of smoke on CFTR

A

Expose to FSK –> cAMP –> CFTR (also Q1)

Can see a reduction in currents when exposed to currents
MEan data - can see that over time CFTR function Is decreasing

46
Q

Effect of smoke on BK channels

A

Use ATP to activated

Can see increase in resposnse to smoke - this is transient - DUE TO THE TRANSIENT NATURE OF CA INCREASE

Means - BK activity is less, and falls with smoke exposire

47
Q

Describe the effect of cigarrate smoke on the ASL

A

Smoke inhibits Cl secretion directly (INHIBITING CFTR) and reduces the driving force (INHIBITING BK) leading to a reduction in mucociliary clearnce

48
Q

What receptors foes smoke activate

A

TGF-B (through TGF-B)

49
Q

What is the effect of activation of TGF-R

A

Phosphorylation of p38 and Smad3

50
Q

What is the effect of p-SMAD3

A

Inhibition of CFTR

51
Q

What is the effect of p-p38

A

Inhibition of Bk

52
Q

What experiments were performed to determine the effect of smoke on the pathways ds of TGF-B

A

Protein expression levels

Increased levels of p-p38, p-psmad3 and p-HSP27

53
Q

What else does p-p38 do (aside from inhibiting BK)

A

Also causes phosphorylation of HSP27

54
Q

What inhibits the p-p38 mediated phosphorylation of HSP27

A

SB202580

55
Q

What inhibits the phosphorylation of p38

A

Pireffenidone

56
Q

What inhibits activation of the TGF-B receptor

A

LY2157299

57
Q

What inhibits the phosphorylation of p-SMAD3

A

SIS3

58
Q

What is the overall effect f smoke (last stage of the pathway)

A

Inhibition of BK and CFTR

Reduction in height of the ASL

59
Q

What should the effect of applying LY be

A

Inhibition of both pathways

60
Q

What was the method of administration of LY…

So what was the control

A

Dissolve in DMSO - vehicle

Vehicle only - DMSO control

61
Q

Describe the data from CFTR when LY applied

A

Inhibition

62
Q

Describe the BK data when Ly applied

A

Inhibition

63
Q

What is not conclusive from the antagonist studies

A

There is not full inhibition so must be some other pathways which have yet to been identified

64
Q

What is the effect of applying LY on the height of the ASL

A

Drops when exposed to smoke

BUT LESS OF A DROP WHEN THE ANTAGONIST, LY IS APPLIED

65
Q

What would be used to block SMAD3

A

SIS3

66
Q

What is the effect of SIS3 (blocking SMAD3) on CFTR and BK

A

No effect on BK - wrong side of the pathway

Relife on some inhibition on CFTR

67
Q

What is the effect of SIS3 on the ASL height

A

Drop in the height of the ASL when apply SIS3

68
Q

What may be used to inhibit p38

A

Pireffenidone

69
Q

What is the effect of P38 inhibition on CFTR and Bk

A

No effect on CFTR (wring side of the pathway)

Relife of inhibition of Bk

70
Q

What is the effect of p38 inhibition on the height of the ALS

A

Blocks some of the loss of the ASL

71
Q

What is the effect of applyinig SB

A

No effect on CFTr (wrong side of the pathway)

Relife of inhibition of BK

72
Q

What is seen wrt the ASL when SB is applied

A

Some recovery of the ASL

73
Q

Is this the only pathway?

A

No there MUST be others involved

BMS346 - Epithelial Physiology (10 decks)

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