Pharmacological Modulation Of Katp Channels

Question 1

What type of channel is a Katp channel?

Answer 1

Inward rectifying K+ channel

Question 2

What does inward rectifying mean? How does it apply to the Katp channel

Answer 2

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

Question 3

Why is influx favoured over efflux for Katp channels?

Answer 3

Efflux is blocked by intracellular contents such as polyamines e.g. Spermine

Question 4

What are polyamines?

Answer 4

Cytoplasmic long chain aliphatic compounds with more than one amine group
The positive charge permits binding to proteins

Question 5

What subunits form the Katp channel?

Answer 5

4 pore forming regions (Kir)

4 regulatory subunits which are sensitive to atp and therefore metabolic state of cell

Question 6

Structure of Kir6.1 and 6.2 subunit?

Answer 6

Intracellular N terminus
TM1
P loop
TM2
Intracellular c terminus

Question 7

Are Kir channels voltage sensitive?

Answer 7

No

Question 8

What associates with Kir subunits?

Answer 8

Sulphonylurea receptor subunit

Regulatory subunit

Question 9

What is the structure of the SUR?

Answer 9

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

Question 10

Where is the important interaction for Kir 6.2 and SUR?

Answer 10

Cytoplasmic N terminus of kir6.2 and TMD0 and L0 linker important for channel gating by SUR

Question 11

Name some drugs which bind to the sulphonylurea regulatory subunit

Answer 11

Gliclazide
Tolbutamide
Glibenclamide

Question 12

What hypoglycaemic drugs act at Katp channels?

Answer 12

Sulphonylureas

Meglitinides

Question 13

ADRS of sulphonylureas

Answer 13

Manly weight gain and hypoglycaemia

Question 14

What do sulphonylureas do at the Katp channel?

Answer 14

Block it
I.e. Close the channel causing cell to depolarise and release insulin in the same way rising glucose causes insulin release

Question 15

Where do the 4 pore forming subunits and the 4 regulatory subunits co assemble?

Answer 15

In the ER.

Then inserted into the membrane

Question 16

The channels are sensitive to low levels of intracellular ATP. What happens when ATP binds?

Answer 16

The channel closes

Question 17

What ratio reflects the metabolic state of the cell?

Answer 17

ADP to ATP ratio

Will govern receptor open probability

Question 18

What is the effect of ADP for channel function?

Answer 18

Stimulates channel to remain open

Question 19

Where is the ATP binding site?

Answer 19

Located on both the Kir 6 and SUR subunit

Question 20

What other factors modulate Katp channel activity?

Answer 20

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

Question 21

What gene encodes for Kir 6.1

Answer 21

KCNJ8

Question 22

What gene encodes for Kir 6.2?

Answer 22

KCNJ11

Question 23

What gene encodes for SUR1?

Answer 23

ABCC8

Question 24

What gene encodes for SUR2a and SUR2b?

Answer 24

ABCC9 (alternative splicing)

Question 25

Where is Kir 6.1 found?

Answer 25

Ubiquitous

Question 26

Where is Kir 6.2 found?

Answer 26

Pancreatic beta cells Brain Heart Skeletal muscle

Question 27

Where is SUR1 found?

Answer 27

Pancreatic beta cells

Question 28

Where is SUR 2A and 2B found?

Answer 28

2A Heart and brain | 2B smooth muscle

Question 29

What is the combination of subunits in pancreatic beta cells?

Answer 29

Kir6.2/SUR1

Question 30

What is the combination of subunits in vascular smooth muscle cells?

Answer 30

Kir6.1/SUR2B

Question 31

What do fluorescence studies using FURA2 of pancreatic beta cells show?

Answer 31

Membrane potential in couple to calcium influx | Aguilar-Bryan et al 1999

Question 32

What does sulphonylureas do to membrane potential?

Answer 32

Cause a depolarisation and action potentials

Question 33

Who conducted the study which showed what subunit area in Katp channels regulates sulphonylurea selectivity?

Answer 33

Ashcroft et al 1999

Question 34

What subunit is more selective for tolbutamide?

Answer 34

SUR1>SUR2

Question 35

How can the difference in selectivity be used to find the tolbutamide binding site?

Answer 35

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

Question 36

What areas is essential for tolbutamide binding?

Answer 36

TMD2 (the areas on the 14th and 15th membrane spanning domain and the distal part of ICL7 and proximal ICL8)

Question 37

For modulating Katp channels why do we want a high selectivity for kir6.2/SUR1 relative for other channel subtypes?

Answer 37

To avoid possible cardiac ADRS E.g. Cardiac muscle has kir6.2/SUR2A We want drug which has a greater affinity for beta cells

Question 38

What two conditions arise from Katp channel mutations?

Answer 38

Permanent neonatal diabetes | Hyperinsulinaemic hypoglycaemic of infancy

Question 39

What is permanent neonatal diabetes and what mutations cause it?

Answer 39

``` 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 ```

Question 40

What are the two types of permanent neonatal diabetes?

Answer 40

Mild - caused by mutation in binding site | Severe - caused by a gating mutation

Question 41

What kir6.2 causes mild PND? And it respond to sulphonylureas?

Answer 41

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

Question 42

What mutations causes severe PND?

Answer 42

Mutation in KCNJ11 (Q52R or I296L) | Mutation in ABCC8

Question 43

What condition is severe PND associated with?

Answer 43

Developmental delay, epilepsy and neonatal diabetes (DEND) | Associate with kir6.2 gating mutations nd some ABCC8 mutations

Question 44

What is the effect of severe PDN with kir6.2 mutations on channel function?

Answer 44

Channel is favours open probability independent if ATP concentrations problem with channel gating (not ATP sensitivity) not responsive to sulphonylureas

Question 45

Severe PND caused by SUR1 mutations do what to Katp channel?

Answer 45

Overactive and channels do not responce to glucose metabolism Memebrane remains hyperpolarise and no insulin release

Question 46

Does sulphonylureas work to treat PND caused by SUR1 mutations?

Answer 46

It depends where the mutation in SUR1 is so individual molecular diagnosis is needed

Question 47

Hyperinsulinaemic hypoglycaemic of infancy (HHI) is due to what mutations?

Answer 47

Kir6.2 SUR1 (50%) There's mutations mean channel is not working and thus memebrane releases depolarised and increases insulin release

Question 48

Prevalence of HHI?

Answer 48

1: 50000 1: 3000 in areas if high consanguinity

Question 49

Class 1 HHI is caused by what?

Answer 49

Protein trafficking defect so no channel at membrane | This is severe

Question 50

Class 2 HHI is characterised by what?

Answer 50

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

Question 51

How can we treat class 2 HHI?

Answer 51

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

Question 52

Name some K+ channel openers - why may they be used for cardiovascular disease?

Answer 52

Diazoxide Minoxidil Nicorandil Vascular SM contains kir6.1/SUR2B therefore openers will cause vasodilation

Question 53

What is diazoxide used for and what SUR subunits does it work on?

Answer 53

Hypertensive crisis and HHI | SUR1 and SUR2B

Question 54

ADRS of diazoxide?

Answer 54

Hyperglycaemia Hirsuitism Hypertrichrosis

Question 55

What is nicorandil used for? Name an ADR

Answer 55

Angina (NO Donor + Katp channel opener) | GI ulceration

Question 56

What is another use of Katp channel openers?

Answer 56

Ischaemic preconditioning | Opening K+ channels mimic ischaemia and decreases AP duration and makes them less likely to fire