Lecture 8 Flashcards
What are the main functions of K+ channels?
- Drive Vm to EK (-90mV)
- K+ channels maintain negative Vm in resting cell
- Regulate cell volume
what are the 3 families of K+ channels?
volage gated
inwardly rectifying
two pore
what are the features of voltage gated K channels?
o6 TM domains
oPore region
o4th subunit – voltage sensor
o4 subunits need to come together = 1 channel
what are the features of inwardly rectifying K channels - Kir ?
oOpen – K+ moves out – hyperpolarises membrane potential o2 TM domains oSingle pore region oNot voltage regulated – no sensor o4 subunits = 1 channel
what are the features of the two pore k channel?
o4 TM domains o2 pore regions per subunit o2 subunits = 1 channel oConstitutively active oHelp set resting potential
examples of voltage gated K channels
KCNQ1/KCNE1
- regulated by the beta subunit E2,
KCNA10
- Found in proximal tubule
examples of Ca2+ activated K channel
SK4, BK
Open when intracellular Ca2+ conc goes up – calcium regulated
3 main families – low, mid and high conductance
example of Kir channel
kir1.1 - ROMK in kidney
examples of 2 pore K channel
TWIK-1, TASK-2
what are the 2 K channels found on the basolateral membrane of an upper airway epithelia?
o KvLQT1 – regulated by KCNE3 (beta subunit) – activated by cAMP n
o SK4 – calcium activated – intermediate conductance
what are the features of the BK channel?
Calcium activated
High conductance
Apical membrane
what happens to BK and SK4 if intracellular Ca rises?
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
why are calcium activated chloride channels so important ?
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
why is the mouse model for CF not as severe?
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
why is it important to permeabilise the basolateral membrane when trying to measure K+ mediated currents?
makes it like the basolateral membrane doesn’t exist. means that K+ isn’t just recycled, so can actually measure the currents
what is paxilline?
BK channel blocker
what is the effect of paxilline on the apical membrane ?
calcium activated stimulation of K secretion is massively reduced – tells us that the BK channels are on the apical membrane
what is the effect of paxilline on the basolateral membrane?
no effect - no BK channels on basolateral membrane
what is the impact of knock down of BK channels
knocks down production of BK channels – reduced short circuit current
what is the impact of non-targeting BK?
and how is non-targeting Bk produced?
No significant difference - v similar to control
Non-targeting = scrambled shRNA
What is the effect of paxilline on ASL height and ciliary beat frequency?
paxilline blocks BK channels - seeing whether BK channels impact on the ASL
Height of ASL reduced, cilia are bent, ciliary beat frequency reduced
What is the effect of a knock down on BK ?
Cant set optimum height of ASL, complete inhibition of cilia function
what is the impact of smoking on CFTR and BK?
CFTR and BK activity falls - Overall less Cl secretion, meaning reduced height of ASL - reduced mucocilliary clearance
how does smoking effect the pathway to cause a reduction in ASL
- 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
What agonists can be used against smoking and what do they target ?
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
impact of LY2157299?
blocks TGF-R receptor = blocks both pathways
Some reversal inhibition of both CFTR & BK.
Some recovery ASL.
impact of SIS3?
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
impact of Pir & SB ?
target p38 and HSP27 - prevent smoke induced inhibition of BK
Some reversal inhibition of BK but not CFTR.
Some recovery ASL.
Patient has a gain of function in their upper airway BK channel. What symptoms might be observed and why?
- 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
