K channels and epithelial function Flashcards
What do K channels do
Open K channels drive the Vm to Ek (Ek = around 90mV) K channels maintain a negative Vm
The more negative the Vm the bigger the driving force to drive chloride secretion - therefore one way to increase chloride secretion is to activate K channels
Characterise the votage gated Kv
K channels where the Po changes with Vm
Made up of 4 subunits each with 6 transmembrane spanning domains which are all required to produce a functional channel
Characterise the inwardly rectifying Kir channel
Two TMD
Again 4 subunits required for one functional channel
Characterise the two pore K channel
They have 4TMDs per subunit
unlike the other subunits in the other channels, the two pore channels have two sequences per subunit that forms the functional pore (the other channels only have one per subunit)
These tend to be constitutively active so are not of much interest
What is chromanol 293B
Pharmacological tool in research that inhibits the action of KCNQ1 (gene) / KVLQT1 (channel protein)
Look at the impact of 293B which gives an idea if the KVLQT1 channel is playing a role in the cell. However it is not the only channel that it effects so is not a conclusive method.
What was the result of RT-PCR of RNA for KvLQT1 in nasal polyps in CF, non CF and HBE cells.
minus lane is a control
KvLQT1 is found in both non-CF and CF patients
KvLQT1 mRNA is expressed in upper respiratory tract epithelial cells with no difference between CF and non CF tissue.
Does KvLQT1 drive Cl secretion? and how was this investigated?
In the presence of amiloride (blocking ENaC so there’s no contamination - Just chloride secretion)
Ussing chamber experiment using nasal epithelium all in the presence of IBMX and forskolin which activate CFTR and KvLQT1 (as they’re activated by cAMP)
The transepithelial potential was measured over time with increasing concentrations of chromanol 293B - This resulted in a reduction in the Vte as conc was increased - i.e blocking KvLQT1 inhibits the secretion of Cl.
How through Ussing chamber work were they sure that the changes in Vte were due to Cl secretion, with no effect from K
Ussing chamber work measures the net fluxes across the epithelium
K ions recycle across the apical and basolateral membranes giving a net flux of essentially zero.
Therefore (with amiloride presence) the only change in Vte is due to the secretion of chloride.
How does chromanol 293B effect ISC/ISC max
As conc increases the size of the current goes down as chloride driving force is reduced.
At 100uM there is no further inhibition meaning there is some chloride secretion still going on not by KvLQT1
The addition of Barium (blocks all K channels) brings the current down to zero. Therefore there are two K channels, KvLQT1 and ?
What is meant by chromanol 293B short ciruit current
Gives a value depicting how much the current has changed by due to 293B
What can be concluded from the graphs below
Left graph:
Non CF tissue results show that stimulation of both Q1 and CFTR increase the response to 293B as there is more chloride secretion.
The CF tissue sees no increase as there is no CFTR function, therefore no Cl secretion so the Q1 block has no effect on Cl current.
Right graph:
Non CF tissue shows 10uA of Cl secretion is driven by the Ba sensitive K channel, this increases upon IBMX stim
CF tissue still has Cl secretion it’s just not mediated by CFTR. The chloride secretion through the non CFTR pathway is being driven by the barium sensitive channel pathway.
Conclude: The Q1 K channel drives Cl secretion through CFTR
The other K channel drives Cl secretion through a different population of Cl channels.
Draw the cell model for the upper airway epithelium
What is hSK4
Medium conductance activated potassium channel which is blocked by clotrimazole
What is CaCC
Calcium activated Cl channel
physiologicaly Ca increase occurs by purinoceptors which are activated by nucleotides such as UTP. These act as ion channels themeselves, allowing calcium into the cell. Or are GPCRs which again lead to an increase in intracellular calcium all leading to an increase in activation of CaCCs
What was the effect of apical UTP on normal and CF tissue on Vte
Addition of UTP stimulates the purinoceptor increasing intracellular calcium. Subsequently there is activation of CaCC giving a hyperpolarising shift in Vte (i.e chloride secretion)
What is the effect on the UTP induced Isc (the amount that the current has gone up by) a measure of chloride secretion.
In normal tissue there is an increase in around 27uA due to activation of CaCC
CF tissue saw a doubling in this response. May be due to an upregulation of CaCC channels which attempt to compensate for the lack of functional CFTR.
Which channels drive Cl secretion through CaCC and how was this shown
UTP induced Isc investigated again in the presence of amiloride but with no cAMP stim. So would expect the activity of Q1 and CFTR to be low, so not much of an effect by chromanol 293B.
Would predict that hSK4 channels and CaCC channels are still active. Addition of clotrimazole results in no Cl secretion.
This means that the UTP activates hSK4 and CaCC. Blocking of hSK4 blocks the driving force for Cl secretion by CaCC is lost.
How does UTP and cAMP stimulation of CF tissue compare to normal tissue upon addition of 293B/Clotrimazole
The control UTP induced current is much higher in the CF tissue due to the compensatory action of CaCC in response to no CFTR. Addition of 293B does reduce the amount of Cl secretion - thought to be as there is no functional CFTR for Q1 to regulate, it switches its role within the cell to stimulate the function of CaCCs in the presence of cAMP
The addition of Clotrimazole significantly reduces Cl secretion - again this is due to the removal of the driving force by a lack of hSK4 function, so no CaCC function either.
What differs between the chloride secretion pathways in healthy and CF tissue
In WT tissue they are clearly paired up: The cAMP pathway and the calcium pathway.
In CF tissue the Q1 channels switch and helps the secretion of Cl through CaCCs
What is the BK channel
The apical Ca activated K channel
What can block the function of BK
Paxilline which is used to show BK function
What is the effect of paxilline on chloride secretion
Pretreatment of paxilline on the apical membrane is significant enough to reduce chloride secretion - this is shown as a reduction in ATP activated Isc.
What is the effect of knocking down BK with shRNA
Less chloride secretion
What physiological role does BK play in cilia beat frequency and how was this investigated?
Cultured upper airway cells and put a liquid layer on the top which either contains just liquid or liquid with paxilline and leave them for 3.5 days. Then measured cilia beat frequency.
Following this they added extra medium with no paxilline into both conditions and again measued beat frequency.
Blocking of the BK channels led to a drop in beat frequency in cilia. Addition of fresh media without paxilline restored normal cilia beating frequency.
Similarly, knocked down BK sees no cilia function. Artificial addition of the ASL restores normal function.
BK IS IMPORTANT FOR CL SECRETION
