Lecture 4 Flashcards
What is the role of the beta subunit
They regulate ion channels - there are a variety of beta subunits that regulate the alpha subunit of the ion channel. They regulate how the channel gates, its voltage dependence, and is capable of changing its trafficking.
Which channels have beta subunits that regulate their activity
Cav, Kv, Kir, Cl- channels
Outline the steps in the cardiac AP and what channels are responsible for each stage.
Depolarisation is due to the opening of Nav channels, followed by the plateau phase due to the opening of calcium channels, the repolarisation phase is due to the opening of Kv channels causing an efflux of potassium. There are multiple Kv channels responsible for the repolarisation phase
What is the result of a mutation in Kv channels in the cardiac myocytes
Repolarisation is slower - prolonging QT
What is the Ikr current and which channel is responsible for it
KCNH2 - action leads to rapid activation/inactivation
It is an inwardly rectifying channel (a consequence of voltage dependence of Po) Also is a delayed rectifier, compared to Nav channels they are quite slow
What part of repolarisation is the Ikr current and the KCNH2 channel responsible for
Early stage of repolarisation
What is the Iks current and what channel is responsible for it
KCNQ1 - Slow current, outward and delayed rectification
What is KCNE1
A family of beta subunits
1 TMD
Regulate Kv channels
How were KCNE1 beta subunits discovered to not be a channel
Once discovered, they overexpressed the subunit in the xenopus oocyte and measured the potassium current. Went from having small to extremely large K current - turns out this was due to KCNE1 upregulating KCNQ1 which was already in the cell.
Can KCNE1 evoke a current by itself
No
Can KCNQ1 evoke a current by itself
Yes but only a very small one
What is the result of co-expressing KCNE1 with KCNQ1
Produce a much greater current at the same driving force. It also slows down the activation time of KCNQ1. The complex formed makes the Iks current which is significantly important for cardiac myocyte repolarisation
Whatis the effect of KCNE1 on KCNQ1 voltage dependence and how was this shown
Normalise the currents from the KCNQ1 channel and plotted an IV curve - Did the same for KCNE1/Q1 and the curve shits to the right. This means that the channel must undergo more depolarisation to achieve the same current relative to KCNQ1 alone.
What is the result of the change in voltage dependence due to the co-expression of KCNE1/Q1
It slows the repolarisation phase and gives the plateau phase of the cardiac action potential
What is the normal resting QT interval time
0.36 seconds
What is the QT interval in a long QT patient
Around 0.54s
What is the effect of the KCNE1 V47F mutant
Difference in time dependence
What is the effect of the KCNE1 L51H mutant
Looks very similar to just KCNQ1 alone
What is the effect of the D76N mutation on KCNQ1 current
Abolishes the current completely in some studies, massively reduced in others.
What is the effect of the W87R mutation on KCNQ1 current
Difference in time dependence compared to WT
What is KCNE1 also known as
MinK
What are three possibilities as to why KCNQ1/KCNE1 currents are significantly reduced.
1 - Gating, e.g. time dependence changes
2 - Trafficking impacts on the number of channels at the membrane
3 - KCNQ1 is regulated by cAMP as a consequence of activation of the B2 adrenergic receptors by the sympathetic nervous system.
How was the trafficking of KCNE1 investigated and which mutant was compared to WT
L51H mutant - Overexpress KCNE1 and tagged it with FLAG. Addition of the FLAG antibody visualised where the KCNE1 protein was located. This was done in permeablised and non-permeablised cells.
What staining could be seen in the L51H mutant KCNE1 in non permeablised cells compared to WT
The L51H mutant showed no staining compared to the WT which had staining around the periphery of the cell.
What staining was seen in the L51H mutant MinK in permeablised cells compared to WT
The L51H mutant was shown in a high concentration beneath the plasma membrane whereas the WT again was found on the periphery of the cell at the plasma membrane. Suggests a problem with trafficking of the mutant KCNE1 to the plasma membrane.
How were co-localisation studies used to investigate the trafficking of KCNE1 and the L51H mutant
Calnexin (a resident ER protein) expression was compared to KCNE1 expression. In WT there is co-localisation of KCNE1 with calnexin but KCNE1 can also be found at the plasma membrane.
Whereas the L51H mutant is shown to be trapped at the ER, co-localised with calnexin.
Only a small amount can be found in the cytosol.
What effect does the L51H mutant have on KCNQ1 trafficking to the membrane
Cells expressing KCNQ1 alone had a reduced number of channels at the plasma membrane compared to cells where co-expression of KCNE1 and Q1 was present. In cells overexpressing KCNE1 with Q1. Q1 is much more represented at the plasma membrane leading to an increase in K current.
How was trafficking of KCNQ1 trafficking investigated
Same as KCNE1 - FLAG tagged - Again with the L51H mutant KCNQ1 was found to be mostly intacellular and almost none at the plasma membrane.
What control experiment was done to show that the KCNE1 B subunit was only responsible for the trafficking of KCNQ1
Same experiment again but instead used the Kv1.4 channel. The WT/mutant had no impact on Kv1.4 trafficking to the cell membrane.
Which LQT syndrome is the KCNE1 mutation found in
LQT5
What can be concluded about LQT5 patients
Not enough KCNQ1/E1 complexes are present at the plasma membrane due to trafficking problems, therefore the Iks current required for repolarisation is not being generated.
What is the effect of sympathetic nervous system stimulation on heart rate
Noradrenaline acts on B2 adrenoreceptors in the heart by G protein coupled receptors (Gs pathway) leading to an increase in cAMP. cAMP activates Iks and increases the current density by slowing inactivation of K channels - accelerating repolarisation and increasing heart rate.
What is the KCNE1/Q1 complex responsible for in exercise and why can LQT5 be lethal when taking part in exercise
Responsible for producing the Iks current which mediates the increased heart rate response when undergoing exercise. Because repolarisation in LQT5 patients is unlikely to become quicker due to trafficking etc - QT unable to keep up with the rest of the heart increasing the risk of arrhythmia and sudden death.
What is Yotiao and what is its role
It is an A-cap regulating protein, it allows PKA to phosphorylate the KCNE1/KCNQ1 complex which keeps them longer for longer.
How is the phosphate on phosphorylated KCNE1/Q1 complex removed
PPI (protein phosphatase type I)
How was the effect of cAMP and Okadaic acid (a PP1 inhibitor) addition to KCNQ1 and KCNQ1/E! complex’s investigated and what were the results
When there is just KCNQ1, addition of cAMP and OA had very little effect on Iks current size. Whereas when the KCNE1/Q1 complex is present there is a much greater magnitude of Iks.
Can conclude KCNE1 is mediating cAMP dependence of KCNQ1.
What KCNE1 mutants were used to investigate KCNE1 induced cAMP dependence of KCNQ1.
W87R and D76N
What was the result on cAMP dependence of KCNQ1 as a result of the W87R KCNE1 mutation
Activation higher in the presence of cAMP, no impact on regulation, fewer channels at the membrane (defined role in trafficking) so the current is slightly less than WT
What was the result on cAMP dependence of KCNQ1 as a result of the D76N KCNE1 mutation
Small K current, no significant difference with or without the addition of cAMP
What problems do patients with the KCNE1 D76N mutant have
Lost the ability to respond to cAMP and therefore the SNS when an increase in heart rate is required. Therefore they are at significant risk of sudden cardiac death when an increase is required. Also have few channels at the plasma membrane.
