Lecture 8

Question 1

What are the main functions of K+ channels?

Answer 1

• Drive Vm to EK (-90mV)
• K+ channels maintain negative Vm in resting cell
• Regulate cell volume

Question 2

what are the 3 families of K+ channels?

Answer 2

volage gated
inwardly rectifying
two pore

Question 3

what are the features of voltage gated K channels?

Answer 3

o6 TM domains
oPore region
o4th subunit – voltage sensor
o4 subunits need to come together = 1 channel

Question 4

what are the features of inwardly rectifying K channels - Kir ?

Answer 4

oOpen – K+ moves out – hyperpolarises membrane potential 
o2 TM domains 
oSingle pore region
oNot voltage regulated – no sensor 
o4 subunits = 1 channel

Question 5

what are the features of the two pore k channel?

Answer 5

o4 TM domains
o2 pore regions per subunit 
o2 subunits = 1 channel 
oConstitutively active 
oHelp set resting potential

Question 6

examples of voltage gated K channels

Answer 6

KCNQ1/KCNE1
- regulated by the beta subunit E2,
KCNA10
- Found in proximal tubule

Question 7

examples of Ca2+ activated K channel

Answer 7

SK4, BK
Open when intracellular Ca2+ conc goes up – calcium regulated
3 main families – low, mid and high conductance

Question 8

example of Kir channel

Answer 8

kir1.1 - ROMK in kidney

Question 9

examples of 2 pore K channel

Answer 9

TWIK-1, TASK-2

Question 10

what are the 2 K channels found on the basolateral membrane of an upper airway epithelia?

Answer 10

o KvLQT1 – regulated by KCNE3 (beta subunit) – activated by cAMP n
o SK4 – calcium activated – intermediate conductance

Question 11

what are the features of the BK channel?

Answer 11

Calcium activated
High conductance
Apical membrane

Question 12

what happens to BK and SK4 if intracellular Ca rises?

Answer 12

o BK and SK4 activate, hyperpolarise the membrane,
o Also activates calcium activated cl channels – second cl secretory pathway
o More Cl secretion through CFTR

Question 13

why are calcium activated chloride channels so important ?

Answer 13

means that in CF, CFTR mutated, but there is still a small amount of Cl secretion . Calcium activated Cl channels are upregulated, meaning impact of CFTR mutation is not as severe.
This means there is still some mucocilliary clearance

Question 14

why is the mouse model for CF not as severe?

Answer 14

they have no problems with airways as they have massive upregulation of calcium activated chloride channels – so take over role of CFTR = normal ASL height and normal mucociliary clearance

Question 15

why is it important to permeabilise the basolateral membrane when trying to measure K+ mediated currents?

Answer 15

makes it like the basolateral membrane doesn’t exist. means that K+ isn’t just recycled, so can actually measure the currents

Question 16

what is paxilline?

Answer 16

BK channel blocker

Question 17

what is the effect of paxilline on the apical membrane ?

Answer 17

calcium activated stimulation of K secretion is massively reduced – tells us that the BK channels are on the apical membrane

Question 18

what is the effect of paxilline on the basolateral membrane?

Answer 18

no effect - no BK channels on basolateral membrane

Question 19

what is the impact of knock down of BK channels

Answer 19

knocks down production of BK channels – reduced short circuit current

Question 20

what is the impact of non-targeting BK?

and how is non-targeting Bk produced?

Answer 20

No significant difference - v similar to control

Non-targeting = scrambled shRNA

Question 21

What is the effect of paxilline on ASL height and ciliary beat frequency?

Answer 21

paxilline blocks BK channels - seeing whether BK channels impact on the ASL
Height of ASL reduced, cilia are bent, ciliary beat frequency reduced

Question 22

What is the effect of a knock down on BK ?

Answer 22

Cant set optimum height of ASL, complete inhibition of cilia function

Question 23

what is the impact of smoking on CFTR and BK?

Answer 23

CFTR and BK activity falls - Overall less Cl secretion, meaning reduced height of ASL - reduced mucocilliary clearance

Question 24

how does smoking effect the pathway to cause a reduction in ASL

Answer 24

• Smoking stimulate TGF-B
• Activates TGF- R
• Phosphorylation of p38 or smad3
• Smad 3 -> inhibits CFTR
• P38 -> inhibits BK
• Additional pathway off p38 – phosphorylation of HSP27 -> inhibit bK
• Reduction in height of ASL

Question 25

What agonists can be used against smoking and what do they target ?

Answer 25

o LY2157299 – blocks TGF-R receptor = blocks both pathways – prevent smoke induced inhibition of BK and CFTR
o SIS3 - targets smad3 - prevent smoke induced inhibition of CFTR
o Pir & SB - target p38 - prevent smoke induced inhibition of BK

Question 26

impact of LY2157299?

Answer 26

blocks TGF-R receptor = blocks both pathways
Some reversal inhibition of both CFTR & BK.
Some recovery ASL.

Question 27

impact of SIS3?

Answer 27

Blocks smad3 phosphorylation
Some reversal inhibition of CFTR
No effect to BK. 
Some recovery ASL.
Suggests that smad 3 may have a role early on – something else has a bigger impact after

Question 28

impact of Pir & SB ?

Answer 28

target p38 and HSP27 - prevent smoke induced inhibition of BK
Some reversal inhibition of BK but not CFTR.
Some recovery ASL.

Question 29

Patient has a gain of function in their upper airway BK channel. What symptoms might be observed and why?

Answer 29

• Increased frequency of respiratory tract infections
• Nasal congestion – repetitive wiping -> sores
• GoF in BK – enhances driving force for Cl secretion (CaCC and CFTR)
• Excess fluid secretion into ASL – increased height
• Mucociliary clearance decreases -> infections and nasal discharge