K channels and epithelial function Flashcards
What do K channels do?
Drive the Vm to Ek
maintain a negative resting membrane potential
regulating cell volume
What are the K channel families
Voltage-gated potassium channel- Kv
Inwardly rectifying potassium channel- Kir
Two pore channel
How many subunits= a functional channel in each K channel family, and how many TMSD do they have?
Voltage gated- 4 subunits= 1 channel, 6 TMSD, 4th domain is the voltage sensor
Kir- 4 subunits= 1 channel, 2 TMSD, no voltage sensor
Two pore- 2 subunits= 1 channel, 4 TMSD
Examples of potassium channels from each family?
KVLQT1
Kir1.1
TWIK1
What is the hypothesis regarding K channel activation and chloride secretion?
K channel activation leads to hyperpolarization of the membrane- more negative resting potential- bigger driving force for Cl secretion
What inhibits KVLQT1? Is it specific
Chromanol 293B
relatively specific- but would need mRNA PCR etc to back up findings
How does chromanol affect the Vte in nasal epithelium?
As more chromanol is added to the basolateral membrane- Vte shifts closer to 0- becomes more positive
block KVLQT1= block Cl secretion
What implied that there were other channels also driving Cl secretion?
Dose response curve- as conc of 293B increased, ISC fell - but it reached a plateau before 0
still some cl secretion
add barium= takes current down to 0
What was the barium sensitive current like for the mutant CF channels?
They showed no response to IBMX
showed barium sensitive Cl secretion
there must be other channels for Cl secretion
What was the first conclusion they made?
Because barium had an effect
there must be other K channels and thus other Cl channels
What other channels did they identify?
HSK4- ca activated K channel
specific blocker= clotrimazole
Cacc- ca activated Cl channel
-activated by UTP- which triggers a rise in IC Ca
How did adding UTP affect the Vte in nasal tissue?
Adding UTP leads to a small and significant hyperpolarising shift in the Vte- signifying Cl secretion- thru ca activated K channels triggering ca activated Cl channels
How did UTP affect ISC in CF tissue?
Leads to a significant increase in UTP induced (ca activated) ISC in the CF tissue
Up-regulation of Cacc in CF
What happens when you use 293B on cells where there is no cAMP stimulation?
The ISC (UTP induced and thus Ca activated current) isn't affected this isnt surprising as no cAMP stimulation= Q1 channels cant function
What happens when you use clotrimazole on cells where there is no cAMP stimulation?
The ISC drops, there is no chloride secretion
calcium stimulated cl secretion needs HSK4 channels to work to provide the driving force for Cl secretion
What happens when you add 293B and clotrimazole in a normal patient? (cAMP stimulation included)
Add 293B- little bit of inhibition- Q1K channels having a role
Add clotrimazole- current drops completely
-driving force for Cl secretion mainly coming from HSK4 channels
What happens when you add 293B and clotrimazole in a CF patient? (cAMP stimulation included)
In control and with 293B- larger currents in CF, they drop slightly with 293B- this is due to enhanced ca activated cl secretion
Add clotrimazole- current completely drops
What was the new thought about K and Cl channels?
Got 2 pairs of potassium and Cl channels
CFTR & Q1
Cacc & HSK4
How does Q1 work in CF cells?
Q1 cells can help drive extra Cl secretion through Cacc
What was discovered about the apical K channel?
BK channel
large K conductance
ca activated
How does paxilline affect BK and how did they know?
Looked at ATP activated Isc
in human bronchial epithelial cells
when they added paxilline to the apical side of the membrane- ISC dropped- cl secretion dropped
if you add paxilline to the basolateral membrane= no effect
What other experi did they do with BK to prove its importance in cl secretion?
Use ShRNA to knockdown BK
looked at ATP induced ISC
reduced the ISC significantly
compared to the not infected and not targeted cells
How does paxilline affect the cilia beat frequency?
The CBF dropped- ASL lower than it should be
proved that it was the ASL affecting the CBF
by manually increasing the ASL in the HBE cells- cbf increases
Whats the conclusion about Bk channels?
Bk channels are essential for chloride secretion and thus the ability of cells to set their ASL layer and thus cbf
