Week 4 physiology (BK - BP and stress)

Question 1

BK channels (acronym)

Answer 1

Big potassium channels

Question 2

mice model in hypertension

Answer 2

• hard to measure systolic/diastolic BP
• measures MAP

Question 3

main determinants of MAP

Answer 3

peripheral resistance
blood volume
cardiac output

Question 4

body responses to blood Volume increase!

Answer 4

2 ways: slow (renal) and fast (cardiovascular)
- excretion via urine
- decreased peripheral resistance and cardiac output.

Question 5

What is a BK channel?

Answer 5

• 6TM (domains):
• Large conductance - transport K+ at a high rate.
• highly specific to K+
• dependent on voltage and intracellular free Ca2+

Question 6

what tissues are BK channels present?

Answer 6

All cell membranes

Question 7

KCNMA1

Answer 7

single gene encoding the pore of the BK channel
ubiquitous in all animals.

Question 8

how does a single gene encoded BK channel perform different functions across different tissues?

Answer 8

The channel has additional accessory subunits that alters its function.
- for example changes the threshold of Ca2+ and voltage needed for activation.

Question 9

6TM..6TM(P) acronym

Answer 9

TM: transmembrane protein
TM(P): transmembrane protein with pore

Question 10

VDCC is …

Answer 10

voltage dependent calcium channels

Question 11

BK channel basic mechanism

Answer 11

in response to depolarisation or high intracellular calcium:
Pump potassium out of the cell

Question 12

VDCC basic mechanism

Answer 12

Dependent on voltage - upon depolarisation:
The channel will induce an inward flux of Ca2+

Question 13

BK channel and VDCC in smooth muscle cells (arteriole)

Answer 13

Adjacent in cell membrane: regulates vascular tone
1. due to depolarisation VDCC allows Ca2+ influx
2. along with ICC’s calcium, BK channel will be activated
3. BK channel releases K+ outwards
4. K+ outward flux will lead to hyperpolarisation.

Question 14

BK channel’s feedback to VDCC

Answer 14

As BK channel leads to hyperpolarisation:
1. this limits VDCC’s voltage dependent action
2. reduces Ca2+ influx

Question 15

How BK channel plays a role in vascular tone

Answer 15

In the VASCULAR SMOOTH MUSCLE CELLS
1. The sarcoplasmic reticulum (SR) has ryanodine receptors that will release Ca2+ stores in “calcium sparks” near the membrane
2. calcium sparks will activate BK channels
3. BK channels induce hyperpolarisation
4. negative feedback to VDCC
5. less contraction - vasodilation

Question 16

experiment (current): calcium sparks and BK channels

Answer 16

Voltage clamp - measure current:
calcium sparks induce small transient outward currents of the membrane through BK channels.

Question 17

ICC is …?

Answer 17

intracellular calcium channel

Question 18

ICC function

Answer 18

release calcium from intracellular stores.
- receptors on the endoplasmic reticulum
- sometimes their action is induced by calcium influx by VDCC.

Question 19

ICC location significance in smooth muscle cells

Answer 19

the ER is close to the plasma membrane - more convenient to respond to calcium influx from VDCC.

Question 20

BK calcium source?

Answer 20

ICC (intracellular) and VDCC (extracellular) and RyR release of Ca2+ (vascular smooth muscle cells).

Question 21

How Ryanodine and IbTX (iberiotoxin) controls arterial diameter?

Answer 21

Ryanodine: blocks RyR (ryanodine receptor) - no calcium sparks ——> no BK activation
IbTX: blocks BK channels
both lead to depolarisation of cell membrane.

Question 22

pharmacological occlusion experiment

Answer 22

Test order of 2 drugs (exchangeably):
first ryanodine then IbTX (IbTX has no further effect)
first IbTX then ryanodine (no further effect)
Those 2 drugs are linked

Question 23

genetic KO of BK channel approaches:

Answer 23

no need to knockout all genes of BK channel:
- often just knockout the exons of the pore forming region
- some try knocking out the first exon, but it doesn’t affect the function of other proteins being transcribed (the channel will still be functional)

Question 24

BK channel’s role in outward current

Answer 24

Plays a major role in outward current, BK channel loss will lead to a major decrease in outward current.

Question 25

BK channel and resting membrane potential (of VSMC)

Answer 25

BK channel presence allows **downward spikes** of membrane potential. **absence** of BK channel: resting membrane potential doesn't have the transient downward spikes and an overall **increased resting membrane potential**.

Question 26

loss of BK channel health implication (using models)

Answer 26

In rodent models: BK channel loss leads to **hypertension**.

Question 27

verifying validity of hypertension in mice models

Answer 27

Loss of BK should solely lead to increase in peripheral resistance, not CO: 1. Increased MAP shouldn't be due to **increased activity**. 2. **Heart weight/Body weight**(heart problem) shouldn't change either, which could affect MAP. 3. **Heart rate** did not increase

Question 28

KO of BK channel alpha vs beta1 subunit

Answer 28

Does KO of beta1 affect BK channel function? **Reduces sensitivity of alpha** (pore-forming) subunit to calcium - normal calcium spark will no longer induce hyperpolarisation.

Question 29

beta1 subunit of BK channel

Answer 29

almost exclusive in vascular smooth muscle cells

Question 30

BK channel in adrenal gland

Answer 30

Normal: Highly expressed in the zona glomerulosa, should play a role in (aldosterone release)

Question 31

BK KO and high aldosterone consequence

Answer 31

high aldosterone leads to higher blood volume. if BK channel is KO in all tissues (including the adrenal gland) there will be higher ALDO release. Normally BK channel opposes high K+-induced ALDO secretion.

Question 32

BK channel expression in HPA axis

Answer 32

1. sparsely expressed in the hypothalamus 2. Highly expressed in the anterior pituitary 3. expressed in zona glomerulosa

Question 33

(BK KO) acute stress effect on plasma ACTH and CORT

Answer 33

BK KO mice show a **reduced increase of plasma ACTH** and **CORT**. Lack of BK channels: **reduced stimulated ACTH release** from pituitary. proves that BK channel is need for an **efficient stress response**. note: CORT will persist for a while even during recovery, but ACTH will drop back

Question 34

anterior pit. corticotrophs identification

Answer 34

**Pomc-GFP** (green fluorescent p.) **mice**. Pomc encodes for ACTH. so the cells in the **anterior pituitary that expresses POMC** are green.

Question 35

CRH and AVP effect on electrical activity

Answer 35

CRH: induces bursts (spike of a slightly longer duration) AVP: increases spike frequency

Question 36

spiking and bursting effect in promoting calcium influx (BK channel)

Answer 36

Bursting cells: **better in secreting hormone** **spiking** -- weak Ca2+ secretion (BK channel **absent**) **bursting** -- robuts Ca2+ secretion (BK channel **present**)

Question 37

Shouldn't BK channels assist in repolarisation instead of bursting (corticotrophs)?

Answer 37

Subset of BK channel (BK near), **near L-type Ca2+ channels**. **Preventing depolarisation to a full spike** (full spike needed to activate the rectifier K+) 1. **prevents rapid repolarisation** (from delayed rectifier K+). 2. potential **oscillates**, until a mechanism completely repolarises it.

Question 38

AVP effect on eletrical current frequency (affect by BK KO?)

Answer 38

increase spike frequency by controlling the conductance of other channels in **BK KO: no bursting**, but increased spike frequency

Question 39

computer modulation in electrical bursting and spikes

Answer 39

First model with CRH: The **computer can remove the electrical current caused by BK channels** Without removing the BK channels pharmacologically, the model of CRH minus (BK channel) shows only spiking (no bursting). **BK channel - bursting behaviour**

Question 40

genetic marker to show **BK channel effect** on **CRH/AVP neuron**

Answer 40

Imaging: **Nur77** -> shows PVN neuron activity (in the context of upper stream hypothalamus) In BK KO mice models, there is a **reduced increase in Nur77** after stress. Indicating that **BK contributes to activation of neuron activity** in the hypothalamus in this context.