Potassium Channels and Epithelia Function

Question 1

What do potassium channels do?

Answer 1

Intracellular K+ is high so when the K+ channels open the mV is driven to -90mV
Therefore, K+ channels maintain a negative Vm

Question 2

What is a negative Vm important for?

Answer 2

For the driving force of Na+ reabsorption and Cl secretion

They also help regulate cell volume

Question 3

What are the three K+ channel families?

Answer 3

Voltage gated Kv
Unwardly rectifying Kir
Two pore domain

Question 4

Describe voltage gated K+ channels

Answer 4

4 subunits = 1 channel
Each subunit = 6TMDs
No.4 TMD is the voltage sensor

Question 5

Describe Inwardly rectifying Kir channels

Answer 5

4 subunits = 1 channel
Each subunit = 2 TMDs
No voltage sensor

Question 6

Describe the two pore domain channels

Answer 6

2 subunits = 1 channel
Each subunit = 4TMDs
Each subunit has 2 pore regions

Question 7

List examples of Kv channels

Answer 7

KCNQ1
KCNE1 , E2 and E3
KCNA10
Ca2+ activated K+ channels = SK4, BK

Question 8

List examples of Kir channels

Answer 8

Kir1.1

ROMK (kidney)

Question 9

List examples of 2P channels

Answer 9

TWIK-1

TASK-2

Question 10

What is the hypothesis of Cl- secretion concerning K+ channels?

Answer 10

K+ maintains Cl- secretion
The basolateral K+ channel sets the negative Vm
If activated the membrane hyperpolarises
More negative = increased driving force for Cl- secretion

Question 11

How is it thought K+ channels affect CFTR?

Answer 11

Activating K+ channels causes an increase in Cl- secretion, even if Po stays the same
However K+ channels and CFTR are usually stimulated at the same time

Question 12

What is the name of the KCNQ1 K+ channel (protein)?

Answer 12

KVLQT1

Question 13

What can block KCNQ1?

Answer 13

Chromanol 293B

Question 14

What does real time PCR of RNA show about KCNQ1 mRNA?

Answer 14

It shows that is expressed in the upper respiratory tract cells

Question 15

In Ussing chamber experiments why is IBMX and forskolin added?

Answer 15

To activate Q1 as it is cAMP activated

Question 16

What was the Ussing chamber experiment used for?

Answer 16

Vte was plotted against time

Question 17

What were the results of the the Ussing chamber experiment for K+ channels in nasal epithelium?

Answer 17

Increasing concentrations of chromanol 293B were added
As concentration increases the Vte rises so it is inhibiting transport
ie less Cl- secretion is occuring so Vte will be more positive

Question 18

Why do we measure Cl- secretion when we look at K+ channel function?

Answer 18

K+ is recycled so can’t be measured directly, so we use Cl- secretion as an indirect measurement
But blocking Q1 = we block Cl- secretion
Increasing concentration of 293B blocks SSC

Question 19

What was the maximal inhibition of 293B?

Answer 19

At 10um we see maximal inhibition but some Cl- secretion is still occuring
If we add Ba2+ then Cl- secretion is equal to zero

Question 20

Why do we use chromanol 293B?

Answer 20

The 293B current takes in to consideration how much short circuit current Q1 is responsible for

Question 21

What is the difference between WT and CF patients in their 293B sensitive currents?

Answer 21

WT: cAMP causes an increase in Cl- secretion which is driven by Q1 channels
CF: There is only a small 293B sensitive current which does not increase when cAMP increases
This is because CFTR channels are not functional - so how does Q1 act?

Question 22

What are the conclusions so far from these experiments?

Answer 22

The basolateral K+ channel is KCNQ1 and is regulated by KCNE3
Q1 is cAMP activated
CFTR is also cAMP activated
So adding Fsk or IBMX should cause an increase in Cl- secretion
However, this is not seen in CF patients
However, adding barium had an effect suggesting that other K+ channels and other Cl- channels are also present in the nasal epithelium

Question 23

What is hSK4?

Answer 23

It is a Ca2+ activated K+ channel which has a relatively specific blocker = clotrimazole
Found on the basolateral membrane

Question 24

What is CaCC?

Answer 24

Ca2+ activated Cl- channel
Activated by UTP (purinoreceptor activation causes an increase in intracellular Ca2+)
(UTP can also activate K+ channels)
Found on the apical membrane

Question 25

What is the hypothesis concerning calcium activated K+ channels?

Answer 25

Calcium acivated K+ channels support Cl- secretion

Hsk4 and CaCC are functionally linked through calcium

Question 26

What is the effect of UTP on normal and CF nasal tissue?

Answer 26

UTP increases Cai
Ussing chamber experiment was used -
UTP causes a hyperpolarising shift which is consistent with Cl- secretion through Ca2+ activated Cl- channels and through activating Ca2+ activated K+ channels

Question 27

How is the response to UTP in CF cells different from the WT?

Answer 27

The response to UTP in CF cells is enhanced

i.e. in CF cells UTP increases SSC more by almost double

Question 28

What is seen in humans and mice with CF in terms of their activated Cl- secretion?

Answer 28

It appears to be upregulated
In mice this is so big that they do not actually have lung problems
However, in humans this is not sufficient to replace with Cl- secretion which would normally occur through CFTR
However, patients would be even worse without this upregulation

Question 29

How do we know that Q1 channels are not activated by Ca2+?

Answer 29

When Q1 channel is blocked with 293B and there is high Ca2+ but low cAMP Cl- secretion is not driven