L8 - K Channels and Epithelia Flashcards
What is the effect of K channels opening in the membrane
What is their function
K moves out of the cell and the Vm is driven towards EK
Maintain and negative Vm
What else do K channels have a role in
Cell volume
Describe the voltage gated Kv channel family
4 subunits - 1 channel
Pore region
6 TMD per subunit, number 4 is the voltage sensor
Describe the inwardly rectifying K channel family
4 subunits of 2 TMD must come together to from a functional pore
Hyperpolarise the Vm when open
2 TMD and a pore region
Describe the two pore domain K channel family
4 TMDs and 2 pore regions per subunit
2 subunits form a channel
Consitiutive activation
What do the 2PD K channels have a role in
Important in setting the resting Vm
Give some examples of Kv channels found in epithelial
KCNQ1 - regulated by the Ex subunit
KCNA10 - found in the proximal tubule
Give some examples of Kv channels that are Ca activated
Sk4 and BK
What other classification is there for the SK4 and BK channels
What does this mean?
Classified based on conductance
Low - mid - high
This is the number if ions that flow through the pore per unit time
Example of Kir channel and its location
Kir 1.1 (ROMK) in the kidney
Examples of two pore domain K channels
TWIK-1
TASK-2
Describe the apical membrane of the upper airway cell - including K channels
APICAL Ca activated chloride channel (secretion of Cl) ENaC CFTR BK K channel (K secretion)
Describe the basolateral membrane of the upper airway cell - INCLUDING K CHANNELS
BASOLATERAL SK4 - K channel - K out Na/K ATPase NKCC1 (Na, K, 2Cl cotransporter (all IN) KCNQ1 (KvLQT1) - E3
What is CaCC activated by
Ca
What is SK4 activated by
Ca
What is CFTR activated by
cAMP
What are BK channels activated by
Ca
What is KVLQT1
KCNQ1
What is the beta subunit E or Q
E
What is KCNQ1 activated by
cAMP
How many pathways are their to trigger Cl secretion in upper airway cells
2
One cAMP and one Ca
Describe the primary pathway for Cl secretion - involving CFTR
Increase in cAMP
Opening of KCNQ1 - K secretion
Hyperpolarisation
Driving force for Cl secretion - through CFTR - also activated by cAMP
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
What technique would be used to measure the short circuit current
Ussing Chamber
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
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
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
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
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
What does it mean if there are large ATP activated currents
Then there is a big function of K channels
What is paxilline
Inhibitor of BK channels at the apical membrane
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
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
What were the three experimental conditios of the BK knockdown expt
NI - not infected
NT- using scrambeled shRNA
KD - using targetting shRNA
What were the results of the BK KD
No difference between NI and NT - expected
In knockdown see significantly decreased currrents
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
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
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
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
Describe the BK-CBF data for KD
Same results as before
Impact BK channel function ….
Impact on Cl secretion
Impact on the height of the ASL
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
How was CFTR activity measured
Using the Cl gdt - whole cell currents
How was BK activity measured
Permeabilise the basolateral membrane
Use gluconate solution
K gradient
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
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
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
What receptors foes smoke activate
TGF-B (through TGF-B)
What is the effect of activation of TGF-R
Phosphorylation of p38 and Smad3
What is the effect of p-SMAD3
Inhibition of CFTR
What is the effect of p-p38
Inhibition of Bk
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
What else does p-p38 do (aside from inhibiting BK)
Also causes phosphorylation of HSP27
What inhibits the p-p38 mediated phosphorylation of HSP27
SB202580
What inhibits the phosphorylation of p38
Pireffenidone
What inhibits activation of the TGF-B receptor
LY2157299
What inhibits the phosphorylation of p-SMAD3
SIS3
What is the overall effect f smoke (last stage of the pathway)
Inhibition of BK and CFTR
Reduction in height of the ASL
What should the effect of applying LY be
Inhibition of both pathways
What was the method of administration of LY…
So what was the control
Dissolve in DMSO - vehicle
Vehicle only - DMSO control
Describe the data from CFTR when LY applied
Inhibition
Describe the BK data when Ly applied
Inhibition
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
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
What would be used to block SMAD3
SIS3
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
What is the effect of SIS3 on the ASL height
Drop in the height of the ASL when apply SIS3
What may be used to inhibit p38
Pireffenidone
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
What is the effect of p38 inhibition on the height of the ALS
Blocks some of the loss of the ASL
What is the effect of applyinig SB
No effect on CFTr (wrong side of the pathway)
Relife of inhibition of BK
What is seen wrt the ASL when SB is applied
Some recovery of the ASL
Is this the only pathway?
No there MUST be others involved
