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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
Describe the secondary pathway of Cl secretion
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
26
If CFTR was the only Cl channel in apical membrane … what would we predict in CF Why is this?
Predict when mutant that mucociliary clearance would stop Have CaCC channels which can be upregulated to compensate
27
Why does the mouse not make a good CF model
CaCC are able to take over from the mutant CFTR sufficiently so that Cl secretion and mucociliary clearance is not significantly impaired
28
Describe how the short circuit current due to K movement can be measured
Permeablise the basolateral membrane Apply gluconate (low chloride) - driving force for Cl secretion Can then measure the Isc mediated by K movement
29
Describe what is meant by "looking at ATP activated currents"
Actually measuring CA ACTIVATED K SECRETION | Since ATP activates purine receptors P2Y and P2X --> increase in Ca
30
What does it mean if there are large ATP activated currents
Then there is a big function of K channels
31
What is paxilline
Inhibitor of BK channels at the apical membrane
32
What is seen to Isc upon the addition of paxilline Why
Decrease in currents Decrease Ca activated K secretion since since BK channels on the apical membrane
33
What sort of experiment was used to verify that BK channels are important for ATP activated ISC
Use a knockdown experiement of the BK channels
34
What were the three experimental conditios of the BK knockdown expt
NI - not infected NT- using scrambeled shRNA KD - using targetting shRNA
35
What were the results of the BK KD
No difference between NI and NT - expected | In knockdown see significantly decreased currrents
36
Describe the mechanism for looking at the ciliary beat frequency
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
In the BK - CBF study what was the test and control conditions - how did this address the question
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
What was 'part 2' of the BK - CBF expt Why was this done?
Non targeted and a knockdown Dfferent way of doing the same experiement
39
Describe the BK_CBF data for when paxilline date
Control CBF of 10Hz Then test CBF see decline (since BK blocked, Cl secretion inhibited) Then add PBS - CBF starts to increase
40
Describe the BK-CBF data for KD
Same results as before
41
Impact BK channel function ….
Impact on Cl secretion | Impact on the height of the ASL
42
Describe the impact of cigarette smoke on CFTR and BK channels
Take HBE cells from smokers and non smokers | Culture until fully differentiated - air liquid interface
43
How was CFTR activity measured
Using the Cl gdt - whole cell currents
44
How was BK activity measured
Permeabilise the basolateral membrane Use gluconate solution K gradient
45
Effect of smoke on CFTR
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
Effect of smoke on BK channels
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
Describe the effect of cigarrate smoke on the ASL
Smoke inhibits Cl secretion directly (INHIBITING CFTR) and reduces the driving force (INHIBITING BK) leading to a reduction in mucociliary clearnce
48
What receptors foes smoke activate
TGF-B (through TGF-B)
49
What is the effect of activation of TGF-R
Phosphorylation of p38 and Smad3
50
What is the effect of p-SMAD3
Inhibition of CFTR
51
What is the effect of p-p38
Inhibition of Bk
52
What experiments were performed to determine the effect of smoke on the pathways ds of TGF-B
Protein expression levels Increased levels of p-p38, p-psmad3 and p-HSP27
53
What else does p-p38 do (aside from inhibiting BK)
Also causes phosphorylation of HSP27
54
What inhibits the p-p38 mediated phosphorylation of HSP27
SB202580
55
What inhibits the phosphorylation of p38
Pireffenidone
56
What inhibits activation of the TGF-B receptor
LY2157299
57
What inhibits the phosphorylation of p-SMAD3
SIS3
58
What is the overall effect f smoke (last stage of the pathway)
Inhibition of BK and CFTR Reduction in height of the ASL
59
What should the effect of applying LY be
Inhibition of both pathways
60
What was the method of administration of LY... So what was the control
Dissolve in DMSO - vehicle Vehicle only - DMSO control
61
Describe the data from CFTR when LY applied
Inhibition
62
Describe the BK data when Ly applied
Inhibition
63
What is not conclusive from the antagonist studies
There is not full inhibition so must be some other pathways which have yet to been identified
64
What is the effect of applying LY on the height of the ASL
Drops when exposed to smoke | BUT LESS OF A DROP WHEN THE ANTAGONIST, LY IS APPLIED
65
What would be used to block SMAD3
SIS3
66
What is the effect of SIS3 (blocking SMAD3) on CFTR and BK
No effect on BK - wrong side of the pathway Relife on some inhibition on CFTR
67
What is the effect of SIS3 on the ASL height
Drop in the height of the ASL when apply SIS3
68
What may be used to inhibit p38
Pireffenidone
69
What is the effect of P38 inhibition on CFTR and Bk
No effect on CFTR (wring side of the pathway) | Relife of inhibition of Bk
70
What is the effect of p38 inhibition on the height of the ALS
Blocks some of the loss of the ASL
71
What is the effect of applyinig SB
No effect on CFTr (wrong side of the pathway) | Relife of inhibition of BK
72
What is seen wrt the ASL when SB is applied
Some recovery of the ASL
73
Is this the only pathway?
No there MUST be others involved

BMS346 - Epithelial Physiology (10 decks)

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