Lecture 7 K Channels and Epithelia

Question 1

How can K+ channels be used to stimulate Cl- secretion in the upper airways

Answer 1

A negative membrane potential in the upper airways will drive Cl- secretion and opening K+ channels has exactly this effect. Opening K+ channels shifts the membrane potential in the hyperpolarising direction shifting the membrane potential more negative towards EK. This increases the driving force for Cl- secretion

Question 2

Other than enhancing the driving force for Cl- secretion what additional role do K+ channels have in epithelia

Answer 2

K+ channels in epithelia play an important role in regulating cell volume

Question 3

How many P-loops or pore domains are required to form the functional pore in K+ channels

Answer 3

4 pore loop domains

Question 4

Outline the three classes of K+ channels are the differences in their subunit composition

Answer 4

Voltage-gated or Kv are K+ channels consisting of 4 subunits each with 6 transmembrane domains. The open probability of these channels changes with Vm. Inwardly rectifying or Kir channels are another family of K+ channels consisting of 4 subunits but this time each with 2 transmembrane domains. These generates large inward negative currents. Finally the two pore domain K+ channels consist of 2 subunits each with 4 transmembrane domains with 2 P loops per subunit

Question 5

What is significant about the activity of the two pore K+ channels

Answer 5

They tend to be constitutively active

Question 6

To which subfamily of voltage-gated K+ channels do the SK4 and BK channels belong

Answer 6

Ca2+-activated K+ channels

Question 7

Give some examples of two-pore domain K+ channels

Answer 7

TWIK-1 TASK-2 and TREK-1

Question 8

What is the role of K+ channels in the upper airways

Answer 8

Maintain Cl- secretion

Question 9

KCNQ1 is a K+ channel found on the basolateral membrane of the upper airway cells what pharmacological agent is capable of blocking KCNQ1

Answer 9

Chromanol 293B

Question 10

How can you determine if there are K+ currents in the airway epithelium mediated by KCNQ1

Answer 10

Treat the cells with chromanol 293B to see if this decreases K+ currents or has an effect on the transepithelial potential

Question 11

What is the downside of chromanol 293B in determining KCNQ1 function

Answer 11

Adding 293B allows you to determine if currents mediated by KCNQ1. However it also blocks other K+ channels and therefore the effects of chromanol 293B are not conclusive

Question 12

What technique was used to determine where KCNQ1 was expressed in the airways of healthy patients and those with CF. Describe the results

Answer 12

RT-PCR was carried out in the nasal epithelium from healthy and CF patients as well as in normal human bronchial epithelium (HBE). A band at 738bps corresponding to the KvLQT1 channel was seen in HBE cells as well as in the nasal epithelium of CF and non-CF patients. Hence KCNQ1 mRNA found in upper respiratory epithelium (nasal epithelium are a model of this) and bronchial epithelium and no difference in mRNA levels or expression levels was observed between CF or non-CF patients

Question 13

Other than by KCNE1 how can KCNQ1 be regulated

Answer 13

KCNQ1 is a cAMP-regulated K+ channel. IBMX/Forskolin treatments increase the transepithelial potential

Question 14

What change in the transepithelial potential would correspond to Cl- secretion

Answer 14

A decrease or negative shift in transepithelial potential corresponds to Cl- secretion

Question 15

What happens to the short circuit currents measured in the airway epithelium as a result of increasing concentrations of chromanol 293B

Answer 15

Increasing concentration of chromanol 293B creates a positive shift in transepithelial potential towards zero (0mV)

Question 16

When using an Ussing chamber to measure the effects of chromanol 293B on KCNQ1 what are you actually measuring and why

Answer 16

Ussing chambers can only measure the net movement of ions across an epithelium in order to determine the transepithelial potential. K+ ion transport across the airway epithelium isn’t a net transport because K+ brought into the cell recycles over both the apical and basolateral membranes. Therefore what Ussing chambers actually measure in this instance is net Cl- secretion although this is driven by K+ channels

Question 17

Below is an concentration-current curve showing the ISC at different concentrations of the KCNQ1 blocker chromanol 293B. Describe what this trace shows

Answer 17

This trace shows that the ISC also goes down with increasing 293B concentration. This corresponds with the idea that smaller K+ are recorded as the amount of KCNQ1 blocker is increased. However this decrease in ISC is only seen up until 10μM. At 10μM 293B further increases in chromanol 293B concentration wont decrease ISC any anymore. This implies there is another K+ channel also driving short circuit currents

Question 18

What type of additional K+ channel was found to be mediating some of the Cl- secretion in the airway epithelium how was this eluded to

Answer 18

Adding Ba2+ to the extracellular solution of the cells treated with chromanol 293B decreased the ISC value from where it was with maximum 293B concentrations to 0. Hence a Ba2+-sensitive channel must also mediate Cl- secretion

Question 19

What is the feature of the current labelled with the red bars what can this be used as an indication of experimentally

Answer 19

The red bar indicates the chromanol 293B-sensitive K+ current mediated by KCNQ1. As K+ currents set the dirving force for Cl- secretion it acts as an indication of the magnitude of the activity of CFTR in Cl- secretion

Question 20

Below is some data on the chromanol 293B-sensitive currents in the nasal epithelium of healthy patients and CF patients either in control conditions or treated with forskolin and IBMX. Describe and explain what this data shows

Answer 20

The graph shows that there is a small 293B-sensitive current in healthy patient epithelia under control conditions. This corresponds to small amounts of Cl- secretion. In non-CF tissue the 293B-sensitive current is much larger in the presence of FSK/IBMX. This is because these compounds act to activated CFTR leading to a stimulation of Cl- secretion. In contrast the CF-tissue had almost no 293B-sensitive currents with or without FSK/IBMX. This is most likely due to the fact that these cells possess no functional CFTR

Question 21

Below is some data on the Ba2+-sensitive currents in the nasal epithelium of healthy patients and CF patients either in control conditions or treated with forskolin and IBMX. Describe and explain what this data shows

Answer 21

The graph shows that there is a Ba2+-sensitive current in control conditions corresponding to small amounts of Cl- secretion. In non-CF tissue the Ba2+-sensitive current is a little larger in the presence of FSK/IBMX. This is due to the stimulation of CFTR. However CF-tissue also had Ba2+-sensitive currents in control conditions hence indicating that some some Cl- secretion in these cells is not mediated by CFTR. In the presence of FSK/IMBX there was however no increase the Ba2+-sensitive currents in this CF patient cells. This implies that the channel that isn’t inhibited by chromanol 293B is not cAMP-dependent

Question 22

What is the role of KCNQ1 in the upper airway epithelium

Answer 22

KCNQ1 drives Cl- secretion through CFTR

Question 23

What can be said about the additional channel in the airway epithelium that is not inhibited by chromanol 293B

Answer 23

This channel is a Ba2+–sensitive K+ channel that drives Cl- secretion independent of CFTR and cAMP

Question 24

What is the identity of the basolateral Ca2+-activated K+ channel in the airway epithelium and what are its main properties

Answer 24

hSK4 is the Ca2+-activated K+ channel found in the basolateral membrane of the airway epithelium. This channel is blocked by clotrimazole produces intermediate conductance and is Ba2+ sensitive

Question 25

What is the identity of the apical Ca2+-activated Cl- channel in the airway epithelium and what are its main properties

Answer 25

CaCC as the Ca2+-activated Cl- channel in the apical membrane of the cell. This channel is activated by Ca2+ fluxes that occurs as a result of purinoceptor activation by UTP

Question 26

Give some examples of purinoceptors and how they differ

Answer 26

Purinoceptors are receptors activated by nucleotides that either act as ions (P2X) channels themselves or are GCPRs (P2Y)

Question 27

How was it thought that the additional channels in the airway epithelium mediated Cl- secretion independent of cAMP and CFTR

Answer 27

Activation of the Ba2+ sensitive hSK4 drives additional Cl- secretion by CaCC

Question 28

How are CFTR and KCNQ1 linked

Answer 28

cAMP activates both channels

Question 29

How are CaCC and hSK4 linked

Answer 29

Ca2+ activates both channels

Question 30

What compound can be used to activate Cl- secretion by CaCC

Answer 30

Addition of UTP can be used to stimulate the purinoceptors leading to a rise in intracellular Ca2+. This leads to a hyperpolarising shift in the transepithelial potential consistent with Cl- secretion

Question 31

Below is some data showing the magnitude of UTP sensitive currents in the nasal epithelial cells of healthy patients and those with CF. Describe and explain what this data shows

Answer 31

Addition of UTP to normal nasal tissue causes an increase in Cl- secretion consistent with a 27μA cm-2 shift in the ISC. Hence the size of the UTP-sensitive current mediated by Ca2+-activated Cl- channels is 27μA cm-2. However the response to addition of UTP in CF patients is double wild type generated twice the UTP induced currents. This is thought to be due to that fact that in CF patients there may be an upregulation of CaCC channels. This would help to give compensation for reduced amounts of CFTR activity

Question 32

Use the data below to outline how was it determined which channels were mediating effects of UTP on Cl- secretion independent of cAMP

Answer 32

The magnitude of the UTP-induced currents was measured in cells in which there was no cAMP in order to minimise KCNQ1 and CFTR activity. Then the effect of adding the KCNQ1 blocker chromanol 293B was tested by adding it to the cells. Unsurprisingly there was little to no effect on the currents generated due to no cAMP. Then the cells were tested for hSK4 activity using the known blocker of the channel clotrimazole. The activity of UTP on Cl- secretion was found to be due to the activity of hSK4 as adding the blocker clotrimazole massively decreased the currents generated

Question 33

What does the data below suggest particularly about the activity of KCNQ1 in patients with CF

Answer 33

Compared to healthy patients CF individuals have a greater UTP-sensitive current this is likely to be due to the upregulation of CaCC channels. In addition the size of the UTP-induced chromanol inhibited currents are also greater implying a greater inhibition of Ca2+-activated Cl- secretion. This in turn suggests that in CF KCNQ1 may have switched from solely driving cAMP-dependent Cl- secretion through CFTR to also driving Cl- secretion through CaCC channels

Question 34

On which membrane is hSK4 and CaCC found

Answer 34

CaCC is found on the apical membrane driven by the basolateral hSK4 channel

Question 35

What other nucleotide can be used to activate purinoceptors and raise intracellular Ca2+ levels to trigger Ca2+-dependent Cl- secretion

Answer 35

ATP

Question 36

What is the identity of the apical Ca2+-activated K+ channel present in the apical membrane of the airway epithelium

Answer 36

BK channel

Question 37

What compound blocks the apical Ca2+-activated Cl- channel responsible for driving Ca2+-dependent Cl- secretion

Answer 37

Paxilline

Question 38

What is the effect of blocking the apical BK channel with paxilline

Answer 38

Decreases size of the ATP-sensitive current indicating a smaller Ca2+-dependent Cl- secretion

Question 39

Other than with pharmacological blockers how has the effect of BK K+ channels been investigated

Answer 39

ShRNA targeted knockdown of the BK channel has been investigated to determine the effect on the ATP-sensitive currents. Knockdown of BK protein decreases ATP-activated ISC hence leading to a decreased Cl- secretion

Question 40

What was the effect of inhibiting the BK channel on the cilia beat frequency in cultured airway epithelia

Answer 40

There was a lower ciliary beat frequency and lower ASL height in airway epithelia exposed to paxilline with cilia beat frequency decreasing to half of control levels. This indicates that Cl- secretion driven by BK is required to drive normal beat frequency

Question 41

What was the effect on cilia beat frequency of removing paxillline from the artificial ASL on covering cultured airway cells

Answer 41

Cilia beat frequency returned to wild type levels

Question 42

What was the effect of experimentally increasing the height of the ASL on the cilia beat frequency of cultured airway cells with BK knocked down

Answer 42

Cilia beat frequency returned to wild type levels

Question 43

Which basolateral channel(s) help set the driving force for Cl- secretion in the airways

Answer 43

K+ channels – the cAMP-dependent KCNQ1 and Ca2+-dependent hSK4

Question 44

Which apical channel(s) help set the driving force for Cl- secretion in the airways

Answer 44

K+ channels – the Ca2+-dependent BK channel

Question 45

Complete the upper airway cell model below with the ion channels/transporters present and the ions they transport

Answer 45

See completed diagram below