L8 - K Channels and Epithelial Function Flashcards
What is the role of K channels in respiratory epithelia?
K+ channels maintain negative Vm
Regulate cell volume – help bring volume down
Drive Cl secretion in upper respiratory tract
What are the 3 molecular K channel families?
Voltage gated
Inward rectifying
Two pore
What is the structure of the voltage gated K channels?
6 transmembrane spanning domains
1 pore region
4th transmembrane spanning domain is voltage sensor
4 subunits required to make a functional channel
What is the structure of the inward rectifying K channels?
2 transmembrane spanning domains
1 pore region
4 subunits required to make a functional channel
What is the structure of the two pore K channels?
4 transmembrane spanning domains
2 pore regions
2 subunits required to make a functional channel
Tend to be constitutively active – help set resting membrane potential
What are the 4 example of voltage gated K channels?
KCNQ1 KCNA10 Ca activated - SK4 - BK
What is the KCNQ1 channel regulated by?
Either KCNE1/E2/E3
Where is KCNA10 found?
Proximal tubule
What are the 3 different states of the SK4 channel?
Low conductance, mid conductance and high conductance
What are Ca activated K channels regulated by?
Open probability regulated by membrane potential and intracellular Ca conc
As intracellular Ca increases as does open probability
What is an example of an inward rectifying K channel?
Kir 1.1 (ROMK)
Found in the kidney
What are 2 examples of two pore K channels?
TWIK-1
TASK-2
What 4 channels are found on the basolateral membrane of an upper airway cell?
KvLQT1 – regulated by KCNE3 – activated by cAMP
SK4 – activated by Ca (mid conductance)
Na/K ATPase
NKCC1
What 4 channels are found on the apical membrane of an upper airway cell?
ENaC
CFTR – activated by cAMP
BK – activated by Ca (high conductance)
CaCC – activated by Ca
How do K channels control Cl secretion?
K+ channels maintain Cl- secretion by hyperpolarising Vm
As intracellular Ca rises in the upper airway cells, SK4 and BK activate and hyperpolarise the membrane
- At the same time the Ca activates CaCC (Ca activated Cl channels) – 2nd Cl secretory pathway in apical membrane
- CFTR will also be active – Cl secretion
In CF patients you would predict if they only had CFTR channels – mucous ciliary clearance should effectively stop in these patients - why does it not?
It doesn’t though – it is just massively slowed
This is because we have CaCC – mediate a small amount of Cl secretion
- Impact of CFTR mutations not as severe as we would expect – as small upregulation of CaCC
Why are animal models of CF not perfect?
CF mouse models do not have any upper airway issues
This is because they have massive upregulation of CaCC
- Replaces the role of CFTR –> normal ASL height
How did they look at the affect of ATP activated Isc K channel data in HBE cells?
Basolateral membrane has been permeabilised (like it doesn’t exist) – K can’t recycle
Gluconate solutions – low Cl solutions
K+ gradient –> K secretion –> K+ mediated currents
- High K basolateral
- Low K apically
How does ATP affect K channels?
ATP activates purine receptors (P2Y and P2X) –> increase in Ca –> looks at Ca activated K secretion
What were the results of the affect of ATP activated Isc K channel data in HBE cells?
Added paxilline to apical membrane – BK channel blocker
Ca activated stimulation of K secretion is massively reduced
- Shows BK channels are on apical membrane
No effect when added basolaterally
How did they test the effect of knocking down BK channels?
Looked at the affect of ATP activated Isc K channel data in HBE cells
- NI - not infected
- NT - not targeted
- scrambles shRNA
- KD - knockdown
What were the results of the affect of ATP activated Isc K channel data in HBE cells? - BK knockdown experiments
Used shRNA – reduced number of BK channels present
Isc increased by 20 mA/cm2
- Much less than in non-infected
What is the air liquid interface method of NHBE cells in culture?
Cultured NHBE cells in same media on basolateral side for 3.5 days
- Upper airway cells with no media on apical side
- The cells make their own ASL
How do you use air liquid surface interface method to test cilia beat frequency in test and control cells?
- Cultured NHBE cells in same media on basolateral side for 3.5 days – air liquid surface interface
- Upper airway cells with no media on apical side
- The cells make their own ASL - Measured cilia beat frequency after 3.5 days
- In test cells – in presence of paxilline
- Paxiline is in medium that is removed from apical side
- However not all of it can be removed
- In control cells - Added PBS to apical membrane – restored ASL
- Control experiment to ensure impact related to ASL height
- If BK channels important
- ASL wont go up to optimum level
- Cilia will bend – cilia beat frequency will decrease - Measured cilia beat frequency
What were the results of the cilia beat frequency experiment with paxilline?
Lower cilia beat frequency due to lower ASL
Paxilline on apical surface –> blocks BK –> reduced Cl secretion –> reduced ASL height –> reduced cilia beat frequency
Know it is due to the height of the ASL as if you add PBS the cilia beat frequency increases
Presence of BK channels are important for ASL height and cilia beat frequency
What two things affect the height of the ASL?
Both Cl secretion and K channels
How did they look at CFTR function in smokers vs non-smokers HBE cells?
Ussing chamber using whole cell
Haven’t permeabilised the basolateral membrane
Set Cl gradients
Look at Isc – Cl secretion – CFTR function
How did they look at K channel function in smokers vs non-smokers HBE cells?
Permeabilised basolateral membrane
Gluconate solutions – low Cl
Set K gradients
Looked aT ISC – K secretion – K channel function
How did they test the impact of cigarette smoke on CFTR?
Exposed HBE cells to forskolin
Forskolin –> increases cAMP –> PKA –> CFTR and Q1 activation –> increased driving force for Cl secretion
What is the impact of cigarette smoke on CFTR?
CFTR activity falls with smoke exposure
- More than it falls normally with air exposure
Shown by fall in Isc
How did they test the impact of cigarette smoke on BL?
Exposed HBE cells to ATP
ATP –> purine receptors –> increased intracellular Ca
What is the impact of cigarette smoke on BK?
BK activity falls with smoke exposure
Shown by fall in Isc
What is the impact of cigarette smoke on ASL?
Reduction in CFTR activity –> less Cl secretion
Reduction in BK activity –> reduced driving force for Cl secretion
Impacts height of ASL –> lower ASL height –> reduction in mucociliary clearance
How does smoking inhibit CFTR?
Smoking –> stimulate TGF-beta –> activates TGF receptor –> phosphorylation of smad3 –> inhibits CFTR
How does smoking inhibit BK?
Smoking –> stimulate TGF-beta –> activates TGF receptor –> phosphorylation of p38 or HSP27 –> inhibits BK
What doesthe LY2157229 antagonist do?
Blocks TFG receptor –> blocks both pathways
What doesthe SIS3 antagonist do?
Blocks smad3
What dothe Pirfenidone and SB203580 antagonists do?
Blocks p38
How is TGF receptor activation by smoking inhibited and results?
By LY2157229
Some reversal of inhibition of both CFTR and BK
- Mainly in the first few hours
Some recovery of ASL height
How is smad3 receptor activation by smoking inhibited and results?
By SIS3
Some reversal of inhibition of CFTR but not BK
- Mainly in the first few hours
Some recovery of ASL height
How is P38 and HSP27 receptor activation by smoking inhibited and results?
By Pirfenidone and SB203580
Some reversal of inhibition of BK but not CFTR
- Mainly in the first few hours
Some recovery of ASL height
A patient has a GOF in their upper airway BK channel. What symptoms might be observed and why?
Increased activity –> open K channels –> drive Vm towards Ek –> hyperpolarisation increases driving force for Cl secretion to the already open Cl channels –> excess fluid secretion into ASL –> increase ASL height –> reduction in cilia beat frequency
Airway struggles to clear the mucous
- Increased risk of respiratory tract infection
- Nasal congestion
- Sores on nose and upper lip area due to repetitive wiping
A patient has a GOF in their upper airway BK channel - how might we treat this patient? Why would this be effective?
Paxilline –> block BK channels directly –> reduce driving force for Cl secretion
Treatment should be inhaled to reduce systemic effect
Could also look at drugs that would activated p38 –> reduce BK activity –> reduce symptoms
