L8 - K+ channels Flashcards
what is the main function of K+ in cells?
- drive the cell to a negative resting potential (drive Vm to Ek)
- high IC K+ and low EC K+
- when K+ channels activated, membrane potential becomes more negative - this creates a driving force for Cl- secretion
give the 3 main families of K+ channels in epithelia
- voltage-gated K+ channels (Kv)
- inwardly rectifiying K+ channels (Kir)
- 2 pore domain K+ channels
give some features of voltage-gated K+ channels
- Po changes with membrane potential
- 6TMD and 1 pore (between TMD5-6)per subunit
- 4 subunits make up a functional channel
Give some features of a inwardly rectifying potassium channel
- greater tendency for K+ uptake
- 2TMD and 1 pore per subunit
- 4 subunits make up a functional channel
Give some features of a 2 pore domain potassium channel
- 4 TMD per subunit
- 2 pores per subunit
- 2 subunits make up 1 channel
- are CONSTITUTIVELY ACTIVE - help set resting potential
How many pore sequences to each different types of K+ channels have?
- each channel has 4 pore sequences
what is the hypothesis to do with K+ channels and Cl- secretion?
K+ maintains Cl- secretions, as open K+ channels cause K+ uptake into the cell - causes hyperpolarisation - creates driving force for Cl- secretion via CFTR
what does chromanol 293B inhibit?
inhibits small number of K+ channels, e.g. KCNQ1 gene
- KvLQT1 channel
Is there any difference between the levels of KVLQT1 in normal and CF tissue? how was this shown?
No, there was no difference between mRNA levels expressed in the upper respiratory tract between normal and CF tissues
- shown doing RT-PCR of RNA in HBE cells line
what did experiments using a ussing chamber on nasal epithelial cells show about Cl- secretion and K+?
- When levels of chromanol 293B rise - there is a change is Vte - this shifts more towards 0 which shows that there is a decrease in Cl- secretion
- cannot be due to changes in K+ as ussing chamber doesn’t measure recycling of K+ - only measures net current
- cannot be due to ENaC- as amiloride is added to avoid interference
- size of CFTR currents reduce with increased 293B as there is a reduced driving force for cl- secretion
- but is still some Cl- secretion as it is not all blocked by 293B - must be other channels driving Cl- secretions
What happens when Ba2+ is added in exp with ussing chamber measuring Cl- secretion in nasal epithelia?
- when Ba2+ is added - this blocks most k+ channels - makes graph/current drop to 0 - must be other channels driving Cl- secretion which are inhibited by Ba2+ but not by chromanol 293B
How was the response to chromanol 293B effected in CF tissue compared to WT?
- when IBMX/fsk to WT the response to chromanol 293B was larger than without IBMX.fsk - shows that more Cl- secretion is going on
- In CF tissues - no change in Cl- secretion when 293B added as no CFTR channels are working anyway
How was the response to Ba2+ effected in CF tissue compared to WT?
- In CF tissue there was a bigger change in Isc when Ba2+ was added compared to when 293 B is added
- shows Cl- secretion must be driven by Ba2+ sensitive K+ channels, and that there must be another form of Cl- secretion apart from CFTR- as CFTR is non-functional in CF patients - but is still Cl-secretion occurring
- also no change in Isc to IBMX/fsk - shows secretion is not due to CFTR
- is no sig difference between CF and WT tissues - both have other mechanism of Cl secretion
what is the basolateral k+ channel that drives Cl-secretion through CFTR?
- is KvLQT1 (Q1) with E3 - this is cAMP activated - can drive Cl- secretion through CFTR
- is chromanol 293B sensitive
What is the other K+ channel on the basolateral membrane of epithelila cells which can drive Cl- secretion?
hSK4
- is a Ca2+ activated K+ channel
- is barium sensitive
- is blocked by clotrimazole
what is another Cl- channel on the apical membrane that is responsible for the small amount of Cl- secretion in CFTR patients?
CaCC
- Ca2+ activated Cl channel
- activated by UTP -this increases IC Ca2+
- purinoceptors activate UTP
what Cl- channel is upregulated in CF and how was this shown?
- CaCC is upregulated in CF patients
- Showed as apical UTP increase in ISc was twice as much in CF tissue nasal as in WT tissue - higher Cl- secretion
- when UTP added - is a hyperpolarising shift
- this gives slight compensation for lack of CFTR
- Is Po or number increased?
what is the K+ channel that drives UTP dependent Cl- secretion? how was this shown?
hSK4
- when SK4 was inhibited by clotrimazole there was no/little UTP induced Cl- secretion
- HSK must be providing driving force for Cl- secretion via CaCC channels.
(when 293B added- no change - shows Cl- secretion is not due to Q1)
what can inhibit CA2+ activated Cl- secretion ?
clotrimazole
can Q1 channels switch?
propose that Q1 channels switch in CF and drive Cl- secretion through CaCC not CFTR
is there controversy in effects Q1 has on Cl- secretion in CF?
Yes
- some exp show that addition of chromanol 293B has no change on Isc - Q1 having no effect/ contribution to Cl- secretion in CF
- other exp show there is a reduction in UTP induced current (Cl- secretion) when chromanol 293B is added - suggests Q1 channels switch
- may be due to diffferent cell models or in vivo/ invitro
- could switch only in some pateitns or at different stages of disease progression
- second exp has camp stimulation but exp before does not have camp stimulation
what is the K+ channel which causes K+ secretion on the apical membrane?
- BK
What can block apical K+ channels and what happens when this is applied?
paxilline
- when this is applied - less K+ secretion, lower liquid layer, 50% reduced cilia beat frequency
what happens when artificial medium is added to BK KD cells?
- brings liquid layer up to a normal height
- cilia beat effectively
- normal function
