K channels and epithelial function

Question 1

What do K channels do?

Answer 1

Drive the Vm to Ek
maintain a negative resting membrane potential
regulating cell volume

Question 2

What are the K channel families

Answer 2

Voltage-gated potassium channel- Kv
Inwardly rectifying potassium channel- Kir
Two pore channel

Question 3

How many subunits= a functional channel in each K channel family, and how many TMSD do they have?

Answer 3

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

Question 4

Examples of potassium channels from each family?

Answer 4

KVLQT1
Kir1.1
TWIK1

Question 5

What is the hypothesis regarding K channel activation and chloride secretion?

Answer 5

K channel activation leads to hyperpolarization of the membrane- more negative resting potential- bigger driving force for Cl secretion

Question 6

What inhibits KVLQT1? Is it specific

Answer 6

Chromanol 293B

relatively specific- but would need mRNA PCR etc to back up findings

Question 7

How does chromanol affect the Vte in nasal epithelium?

Answer 7

As more chromanol is added to the basolateral membrane- Vte shifts closer to 0- becomes more positive
block KVLQT1= block Cl secretion

Question 8

What implied that there were other channels also driving Cl secretion?

Answer 8

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

Question 9

What was the barium sensitive current like for the mutant CF channels?

Answer 9

They showed no response to IBMX
showed barium sensitive Cl secretion
there must be other channels for Cl secretion

Question 10

What was the first conclusion they made?

Answer 10

Because barium had an effect

there must be other K channels and thus other Cl channels

Question 11

What other channels did they identify?

Answer 11

HSK4- ca activated K channel
specific blocker= clotrimazole
Cacc- ca activated Cl channel
-activated by UTP- which triggers a rise in IC Ca

Question 12

How did adding UTP affect the Vte in nasal tissue?

Answer 12

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

Question 13

How did UTP affect ISC in CF tissue?

Answer 13

Leads to a significant increase in UTP induced (ca activated) ISC in the CF tissue
Up-regulation of Cacc in CF

Question 14

What happens when you use 293B on cells where there is no cAMP stimulation?

Answer 14

The ISC (UTP induced and thus Ca activated current) isn't affected
this isnt surprising as no cAMP stimulation= Q1 channels cant function

Question 15

What happens when you use clotrimazole on cells where there is no cAMP stimulation?

Answer 15

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

Question 16

What happens when you add 293B and clotrimazole in a normal patient? (cAMP stimulation included)

Answer 16

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

Question 17

What happens when you add 293B and clotrimazole in a CF patient? (cAMP stimulation included)

Answer 17

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

Question 18

What was the new thought about K and Cl channels?

Answer 18

Got 2 pairs of potassium and Cl channels
CFTR & Q1
Cacc & HSK4

Question 19

How does Q1 work in CF cells?

Answer 19

Q1 cells can help drive extra Cl secretion through Cacc

Question 20

What was discovered about the apical K channel?

Answer 20

BK channel
large K conductance
ca activated

Question 21

How does paxilline affect BK and how did they know?

Answer 21

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

Question 22

What other experi did they do with BK to prove its importance in cl secretion?

Answer 22

Use ShRNA to knockdown BK
looked at ATP induced ISC
reduced the ISC significantly
compared to the not infected and not targeted cells

Question 23

How does paxilline affect the cilia beat frequency?

Answer 23

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

Question 24

Whats the conclusion about Bk channels?

Answer 24

Bk channels are essential for chloride secretion and thus the ability of cells to set their ASL layer and thus cbf