L4 - B subunits Flashcards
what are Beta subunits?
small molecular weighted subunits, usually with 1TMD, which interact with the ion channel (alpha subunit) to change and regulate its properties
what happens in the cardiac AP?
- fast influx of Na+ during depolarsation (when -70 Vm thresold reached)
- platueu stage due to slow Ca2+ influx (NA+ decrease)
- repolarisation due to fast K+ efflux (outward rectification) (PCa+ decrease)
what causes K+ efflux in cardiac AP?
Ikr and Iks channels
balance between the 2 channels determines K+ efflux
give the properties of the Ikr channel?
- rapid activation and inactivation (quicker than other K+ channels)
- inward rectifier
- delayed rectifier (slower than sodium channels)
- KCNH1 (Herg1)
give the properties of the Iks channel?
- slow activation (in comparison to Ikr)
- outward rectifier
- delayed rectifier (compared to sodium channels)
- KCNQ1 (Kvlqt1)
what regulates KCNQ1?
-KCNE beta subunit family
KCNQ1 is the alpha subunit which forms an outwardly rectifying potassium channel (heart myocytes).
give the members of the KCNE family (B subunits which regulate KCNQ1 properties) (in heart myocytes)
- KCNE1 (minK)
- KCNE2 Mirp1
- KCNE3 Mirp2
- KCNE4 Mirp3
- KCNE5 Mirp4
why is KCNE 1 called Mink?
it is small (minimal) so why called min
- wasnorgiginally thought to actually be a potassium channel (a subunit) so why called MinK
what effect does KCNE1 have on KCNQ1?
- tested in hamster ovary cells
- if KCNE1 is overexpressed (without KCNQ1) no current is generated as KCNE1 is not a channel.
- if KCNE1 is overexpressed with KCNQ1 a bigger current is recorded but activation is slowed (time delay to reach a steady state)
- rightward SHIFT IN VOLTAGE DEPENDENCE (need a more positive value for Iks channel to activate)
- shows KCNE1 is regulating the activation of KCNQ1 (changes potential at which channels activate)
what condition arises when KCNE1 is mutated?
- long QT syndrome (LQT5)
- higher risk of arrythmias and sudden death
- repolarisation defect/delay
- prolonged QT interval
- apparent during excercise
what is the incidence of LQT?
1 in 10,000 to 1 in 15,000
what happens to the current in mutants which cause LQT5?
There is a smaller current and different time dependencies
what could be causing a smaller current?
- gating issues
- traffiking issues - less of channel at the membrane
- issues in regulation
what KCNE1 mutation effects gating?
L51H
what KCNE1 mutaitons impacts on traffiking and how do they know this?
L51H
- tagged E1 with an antibody FLAG and looked for in permeabilised and non-permeabilised cells
- WT E1 is present at the membrane
- L51 H E1 is not present at the membrane in non-permeabilised cells but it seen in permeabilised cells so shows there is L51H present in the cells - but it is not being traffiked to the membrane.
what is calnexin?
an ER marker (chaperone)
where is WT KCNE1 seen?
in membrane and in ER (marked by overlap with calnexin)
where is L51H found in the cell?
abundant in ER (hardly any/none in the membrane) - stuck in ER - not allowed to leave as incorrectly foled? (CNX cycle)- eventually sent for degradation via the ERAD pathway.
(so not traffiked past the ER)
what does E1 do to Q1 location in the cell?
- When E1 added to Q1 it started traffiking out of the ER (Q1 alone - lots in ER)
- E1 chaperones Q1 to the membrane?
what happens when L51H is added to KCNq1?
dont see much E1 or Q1 at the membrane - lots in ER
why is Iks current smaller when E1 is mutated?
- current is smaller as less Q1 channels are at the membrane due to a beta subunit/chaperone mutation in E1.
- channels cannot traffic to the membrane efficiently
why was Kv1.4 used in conjuction with E1 mutation (L51H)?
used as a control
- when Kv1.4 used, the E1 mutation had no effect on the traffiking of Kv1.4 and Kv1.4 was still seen at the membrane normally.
- this shows that KCNE1 + its mutation is specific to the properties of KCNQ1.
How do mutations in KCNE1 cause LQT syndrome?
- mutaitons in KCNE1 impact on the traffiking og KCNQ1- therefore there is less KCNQ1 at the cell membrane
- this results in smaller Iks currents.
- as there are smaller potassium currents, there is a slower efflux and slower outward rectification of poatssium ions from the heart myocyte.
- This means repolarisation/inactivation takes longer to occur so the QT interval of the heart myocyte is extended beyond a normal range
- this can cause arrythmias and a higher risk of sudden death.
how is Kis regulated by cAMP?
- sympathetic NS - increase heart rate - during exercise
- beta adrenoceptors are activated which stimulate camp (PKA)
- this causes activation of Iks (phosphoylation of Q1 by PKA)
- this slows inactivation (of Iks channel)- get bigger currents
- This increases the repolarisation rate
- this increases heart rate
- PPI removes P group (when don’t need high heart rate)
ocadaic acid inhibits PPI so dont get dephosphorylation - high currents + increased heart rate for longer
what are the subunits alongside Q1?
E1
yotiao - allows PKA to come alongside Q1
- PKA phosphorylates Q1
- PPI dephosphorylates Q1
what does camp dependence of Q1 channel depend on?
E1 needs to also be present
- when ocodaic acid is added - there is little activaiton on Q1 when E1 is not present
what happens in the D76N mutation?
the ability of E1/Q1 to respond to the sympathetic NS is lost
- unable to speed up heart rate during exercise
what additional subunits - apart from E1 may there be alongside Q1?
- yotiao
- 14-3-3
- KChips
- KChap
- KvB
