Potassium Channels and Epithelia Function Flashcards
What do potassium channels do?
Intracellular K+ is high so when the K+ channels open the mV is driven to -90mV
Therefore, K+ channels maintain a negative Vm
What is a negative Vm important for?
For the driving force of Na+ reabsorption and Cl secretion
They also help regulate cell volume
What are the three K+ channel families?
Voltage gated Kv
Unwardly rectifying Kir
Two pore domain
Describe voltage gated K+ channels
4 subunits = 1 channel
Each subunit = 6TMDs
No.4 TMD is the voltage sensor
Describe Inwardly rectifying Kir channels
4 subunits = 1 channel
Each subunit = 2 TMDs
No voltage sensor
Describe the two pore domain channels
2 subunits = 1 channel
Each subunit = 4TMDs
Each subunit has 2 pore regions
List examples of Kv channels
KCNQ1
KCNE1 , E2 and E3
KCNA10
Ca2+ activated K+ channels = SK4, BK
List examples of Kir channels
Kir1.1
ROMK (kidney)
List examples of 2P channels
TWIK-1
TASK-2
What is the hypothesis of Cl- secretion concerning K+ channels?
K+ maintains Cl- secretion
The basolateral K+ channel sets the negative Vm
If activated the membrane hyperpolarises
More negative = increased driving force for Cl- secretion
How is it thought K+ channels affect CFTR?
Activating K+ channels causes an increase in Cl- secretion, even if Po stays the same
However K+ channels and CFTR are usually stimulated at the same time
What is the name of the KCNQ1 K+ channel (protein)?
KVLQT1
What can block KCNQ1?
Chromanol 293B
What does real time PCR of RNA show about KCNQ1 mRNA?
It shows that is expressed in the upper respiratory tract cells
In Ussing chamber experiments why is IBMX and forskolin added?
To activate Q1 as it is cAMP activated
What was the Ussing chamber experiment used for?
Vte was plotted against time
What were the results of the the Ussing chamber experiment for K+ channels in nasal epithelium?
Increasing concentrations of chromanol 293B were added
As concentration increases the Vte rises so it is inhibiting transport
ie less Cl- secretion is occuring so Vte will be more positive
Why do we measure Cl- secretion when we look at K+ channel function?
K+ is recycled so can’t be measured directly, so we use Cl- secretion as an indirect measurement
But blocking Q1 = we block Cl- secretion
Increasing concentration of 293B blocks SSC
What was the maximal inhibition of 293B?
At 10um we see maximal inhibition but some Cl- secretion is still occuring
If we add Ba2+ then Cl- secretion is equal to zero
Why do we use chromanol 293B?
The 293B current takes in to consideration how much short circuit current Q1 is responsible for
What is the difference between WT and CF patients in their 293B sensitive currents?
WT: cAMP causes an increase in Cl- secretion which is driven by Q1 channels
CF: There is only a small 293B sensitive current which does not increase when cAMP increases
This is because CFTR channels are not functional - so how does Q1 act?
What are the conclusions so far from these experiments?
The basolateral K+ channel is KCNQ1 and is regulated by KCNE3
Q1 is cAMP activated
CFTR is also cAMP activated
So adding Fsk or IBMX should cause an increase in Cl- secretion
However, this is not seen in CF patients
However, adding barium had an effect suggesting that other K+ channels and other Cl- channels are also present in the nasal epithelium
What is hSK4?
It is a Ca2+ activated K+ channel which has a relatively specific blocker = clotrimazole
Found on the basolateral membrane
What is CaCC?
Ca2+ activated Cl- channel
Activated by UTP (purinoreceptor activation causes an increase in intracellular Ca2+)
(UTP can also activate K+ channels)
Found on the apical membrane
What is the hypothesis concerning calcium activated K+ channels?
Calcium acivated K+ channels support Cl- secretion
Hsk4 and CaCC are functionally linked through calcium
What is the effect of UTP on normal and CF nasal tissue?
UTP increases Cai
Ussing chamber experiment was used -
UTP causes a hyperpolarising shift which is consistent with Cl- secretion through Ca2+ activated Cl- channels and through activating Ca2+ activated K+ channels
How is the response to UTP in CF cells different from the WT?
The response to UTP in CF cells is enhanced
i.e. in CF cells UTP increases SSC more by almost double
What is seen in humans and mice with CF in terms of their activated Cl- secretion?
It appears to be upregulated
In mice this is so big that they do not actually have lung problems
However, in humans this is not sufficient to replace with Cl- secretion which would normally occur through CFTR
However, patients would be even worse without this upregulation
How do we know that Q1 channels are not activated by Ca2+?
When Q1 channel is blocked with 293B and there is high Ca2+ but low cAMP Cl- secretion is not driven
