Lecture 4

Question 1

What is the role of the beta subunit

Answer 1

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.

Question 2

Which channels have beta subunits that regulate their activity

Answer 2

Cav, Kv, Kir, Cl- channels

Question 3

Outline the steps in the cardiac AP and what channels are responsible for each stage.

Answer 3

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

Question 4

What is the result of a mutation in Kv channels in the cardiac myocytes

Answer 4

Repolarisation is slower - prolonging QT

Question 5

What is the Ikr current and which channel is responsible for it

Answer 5

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

Question 6

What part of repolarisation is the Ikr current and the KCNH2 channel responsible for

Answer 6

Early stage of repolarisation

Question 7

What is the Iks current and what channel is responsible for it

Answer 7

KCNQ1 - Slow current, outward and delayed rectification

Question 8

What is KCNE1

Answer 8

A family of beta subunits
1 TMD
Regulate Kv channels

Question 9

How were KCNE1 beta subunits discovered to not be a channel

Answer 9

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.

Question 10

Can KCNE1 evoke a current by itself

Answer 10

No

Question 11

Can KCNQ1 evoke a current by itself

Answer 11

Yes but only a very small one

Question 12

What is the result of co-expressing KCNE1 with KCNQ1

Answer 12

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

Question 13

Whatis the effect of KCNE1 on KCNQ1 voltage dependence and how was this shown

Answer 13

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.

Question 14

What is the result of the change in voltage dependence due to the co-expression of KCNE1/Q1

Answer 14

It slows the repolarisation phase and gives the plateau phase of the cardiac action potential

Question 15

What is the normal resting QT interval time

Answer 15

0.36 seconds

Question 16

What is the QT interval in a long QT patient

Answer 16

Around 0.54s

Question 17

What is the effect of the KCNE1 V47F mutant

Answer 17

Difference in time dependence

Question 18

What is the effect of the KCNE1 L51H mutant

Answer 18

Looks very similar to just KCNQ1 alone

Question 19

What is the effect of the D76N mutation on KCNQ1 current

Answer 19

Abolishes the current completely in some studies, massively reduced in others.

Question 20

What is the effect of the W87R mutation on KCNQ1 current

Answer 20

Difference in time dependence compared to WT

Question 21

What is KCNE1 also known as

Answer 21

MinK

Question 22

What are three possibilities as to why KCNQ1/KCNE1 currents are significantly reduced.

Answer 22

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.

Question 23

How was the trafficking of KCNE1 investigated and which mutant was compared to WT

Answer 23

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.

Question 24

What staining could be seen in the L51H mutant KCNE1 in non permeablised cells compared to WT

Answer 24

The L51H mutant showed no staining compared to the WT which had staining around the periphery of the cell.

Question 25

What staining was seen in the L51H mutant MinK in permeablised cells compared to WT

Answer 25

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.

Question 26

How were co-localisation studies used to investigate the trafficking of KCNE1 and the L51H mutant

Answer 26

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.

Question 27

What effect does the L51H mutant have on KCNQ1 trafficking to the membrane

Answer 27

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.

Question 28

How was trafficking of KCNQ1 trafficking investigated

Answer 28

Same as KCNE1 - FLAG tagged - Again with the L51H mutant KCNQ1 was found to be mostly intacellular and almost none at the plasma membrane.

Question 29

What control experiment was done to show that the KCNE1 B subunit was only responsible for the trafficking of KCNQ1

Answer 29

Same experiment again but instead used the Kv1.4 channel. The WT/mutant had no impact on Kv1.4 trafficking to the cell membrane.

Question 30

Which LQT syndrome is the KCNE1 mutation found in

Answer 30

LQT5

Question 31

What can be concluded about LQT5 patients

Answer 31

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.

Question 32

What is the effect of sympathetic nervous system stimulation on heart rate

Answer 32

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.

Question 33

What is the KCNE1/Q1 complex responsible for in exercise and why can LQT5 be lethal when taking part in exercise

Answer 33

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.

Question 34

What is Yotiao and what is its role

Answer 34

It is an A-cap regulating protein, it allows PKA to phosphorylate the KCNE1/KCNQ1 complex which keeps them longer for longer.

Question 35

How is the phosphate on phosphorylated KCNE1/Q1 complex removed

Answer 35

PPI (protein phosphatase type I)

Question 36

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

Answer 36

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.

Question 37

What KCNE1 mutants were used to investigate KCNE1 induced cAMP dependence of KCNQ1.

Answer 37

W87R and D76N

Question 38

What was the result on cAMP dependence of KCNQ1 as a result of the W87R KCNE1 mutation

Answer 38

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

Question 39

What was the result on cAMP dependence of KCNQ1 as a result of the D76N KCNE1 mutation

Answer 39

Small K current, no significant difference with or without the addition of cAMP

Question 40

What problems do patients with the KCNE1 D76N mutant have

Answer 40

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.