Pharmacological Modulation Of Katp Channels Flashcards
What type of channel is a Katp channel?
Inward rectifying K+ channel
What does inward rectifying mean? How does it apply to the Katp channel
Favours ion influx over efflux
Voltage current relegation ship does not follow ohms laws. However, since Ek is approx -80mV, at physiological potentials K+ still moves out of cell
Why is influx favoured over efflux for Katp channels?
Efflux is blocked by intracellular contents such as polyamines e.g. Spermine
What are polyamines?
Cytoplasmic long chain aliphatic compounds with more than one amine group
The positive charge permits binding to proteins
What subunits form the Katp channel?
4 pore forming regions (Kir)
4 regulatory subunits which are sensitive to atp and therefore metabolic state of cell
Structure of Kir6.1 and 6.2 subunit?
Intracellular N terminus TM1 P loop TM2 Intracellular c terminus
Are Kir channels voltage sensitive?
No
What associates with Kir subunits?
Sulphonylurea receptor subunit
Regulatory subunit
What is the structure of the SUR?
Receptor subunit split into 3 discrete transmembrane sections consisting (TM0, TM1 and TM2) of 5, 6 and 6 membrane spanning segments respectively
Extracellular N
TM0
Intracellular linker between TM0 and TM1 (L0)
TM1
Intracellular linker between TM1 and TM2 (L1) contains NBD1
TM2
Intracellular C terminus with NBD2
Where is the important interaction for Kir 6.2 and SUR?
Cytoplasmic N terminus of kir6.2 and TMD0 and L0 linker important for channel gating by SUR
Name some drugs which bind to the sulphonylurea regulatory subunit
Gliclazide
Tolbutamide
Glibenclamide
What hypoglycaemic drugs act at Katp channels?
Sulphonylureas
Meglitinides
ADRS of sulphonylureas
Manly weight gain and hypoglycaemia
What do sulphonylureas do at the Katp channel?
Block it
I.e. Close the channel causing cell to depolarise and release insulin in the same way rising glucose causes insulin release
Where do the 4 pore forming subunits and the 4 regulatory subunits co assemble?
In the ER.
Then inserted into the membrane
The channels are sensitive to low levels of intracellular ATP. What happens when ATP binds?
The channel closes
What ratio reflects the metabolic state of the cell?
ADP to ATP ratio
Will govern receptor open probability
What is the effect of ADP for channel function?
Stimulates channel to remain open
Where is the ATP binding site?
Located on both the Kir 6 and SUR subunit
What other factors modulate Katp channel activity?
Level of phosphotidylinositol 4,5 bisphosphate
Increase in PIP2 increases activity (increases open probability and decreases ATP sensitivity) therefore contents of memebrane important. PIP2 cleavage by PLC will regulate channel activity
Acidic intracellular environment also stimulates activity
What gene encodes for Kir 6.1
KCNJ8
What gene encodes for Kir 6.2?
KCNJ11
What gene encodes for SUR1?
ABCC8
What gene encodes for SUR2a and SUR2b?
ABCC9 (alternative splicing)
Where is Kir 6.1 found?
Ubiquitous
Where is Kir 6.2 found?
Pancreatic beta cells
Brain
Heart
Skeletal muscle
Where is SUR1 found?
Pancreatic beta cells
Where is SUR 2A and 2B found?
2A Heart and brain
2B smooth muscle
What is the combination of subunits in pancreatic beta cells?
Kir6.2/SUR1
What is the combination of subunits in vascular smooth muscle cells?
Kir6.1/SUR2B
What do fluorescence studies using FURA2 of pancreatic beta cells show?
Membrane potential in couple to calcium influx
Aguilar-Bryan et al 1999
What does sulphonylureas do to membrane potential?
Cause a depolarisation and action potentials
Who conducted the study which showed what subunit area in Katp channels regulates sulphonylurea selectivity?
Ashcroft et al 1999
What subunit is more selective for tolbutamide?
SUR1>SUR2
How can the difference in selectivity be used to find the tolbutamide binding site?
Create chimeric proteins through joining genes for SUR1 and SUR2. Creates a protein with functional properties derived from each original protein.
Since SUR2 tolbutamide selectivity is weaker than SUR1 a chimer where the SUR2 subunit section replaces the respective SUR1 subunit section for which tolbutamide binds will show an increase in conductance. This is because it is not blocked by tolbutamide.
The opposite could be done whereby SUR1 subunit section inserted into SUR2 subunit at area where tolbutamide will bind will show an increased selectivity to tolbutamide
What areas is essential for tolbutamide binding?
TMD2 (the areas on the 14th and 15th membrane spanning domain and the distal part of ICL7 and proximal ICL8)
For modulating Katp channels why do we want a high selectivity for kir6.2/SUR1 relative for other channel subtypes?
To avoid possible cardiac ADRS
E.g. Cardiac muscle has kir6.2/SUR2A
We want drug which has a greater affinity for beta cells
What two conditions arise from Katp channel mutations?
Permanent neonatal diabetes
Hyperinsulinaemic hypoglycaemic of infancy
What is permanent neonatal diabetes and what mutations cause it?
Diabetes diagnosed within 6 months of birthed that does not resolve 1:260000 levels births 30% Kir6.2 20% SUR1 50% other e.g. Glucokinass
What are the two types of permanent neonatal diabetes?
Mild - caused by mutation in binding site
Severe - caused by a gating mutation
What kir6.2 causes mild PND? And it respond to sulphonylureas?
R201H/C mutation in ATP binding site
Decreases sensitivity to ATP (more atp needed to decrease channel conductance)
Channel favours open state
Yes it does respond to sulphonylureas
What mutations causes severe PND?
Mutation in KCNJ11 (Q52R or I296L)
Mutation in ABCC8
What condition is severe PND associated with?
Developmental delay, epilepsy and neonatal diabetes (DEND)
Associate with kir6.2 gating mutations nd some ABCC8 mutations
What is the effect of severe PDN with kir6.2 mutations on channel function?
Channel is favours open probability independent if ATP concentrations problem with channel gating (not ATP sensitivity) not responsive to sulphonylureas
Severe PND caused by SUR1 mutations do what to Katp channel?
Overactive and channels do not responce to glucose metabolism
Memebrane remains hyperpolarise and no insulin release
Does sulphonylureas work to treat PND caused by SUR1 mutations?
It depends where the mutation in SUR1 is so individual molecular diagnosis is needed
Hyperinsulinaemic hypoglycaemic of infancy (HHI) is due to what mutations?
Kir6.2
SUR1 (50%)
There’s mutations mean channel is not working and thus memebrane releases depolarised and increases insulin release
Prevalence of HHI?
1: 50000
1: 3000 in areas if high consanguinity
Class 1 HHI is caused by what?
Protein trafficking defect so no channel at membrane
This is severe
Class 2 HHI is characterised by what?
Functional channel mutation (normally in SUR1) which causes channel to remain closed and decreased effect of ADP to open channel
It is mild in nature
How can we treat class 2 HHI?
K+ channel openers - diazoxide
However, to treat a functional Katp channel is needed
Mutations which cause structural channel changes may not be as responsive to diazoxide
Name some K+ channel openers - why may they be used for cardiovascular disease?
Diazoxide
Minoxidil
Nicorandil
Vascular SM contains kir6.1/SUR2B therefore openers will cause vasodilation
What is diazoxide used for and what SUR subunits does it work on?
Hypertensive crisis and HHI
SUR1 and SUR2B
ADRS of diazoxide?
Hyperglycaemia
Hirsuitism
Hypertrichrosis
What is nicorandil used for? Name an ADR
Angina (NO Donor + Katp channel opener)
GI ulceration
What is another use of Katp channel openers?
Ischaemic preconditioning
Opening K+ channels mimic ischaemia and decreases AP duration and makes them less likely to fire
