Lecture 10 - Beta Subunit interactions

Question 1

Beta Subunits

Answer 1

are proteins that don’t act as an ion channel but modify properties of alpha subunits which act as the ion channel

• Important for normal epithelial function
• E.g KCNE and Barttin

Question 2

KCNE Family

Answer 2

KCNE1- 103 to 177 amino acids
KCNE2 - 1 TM domain
KCNE3 - Excitable - Long QT syndrome
KCNQ1- Long QT syndrome

Question 3

Varying effect on function

Answer 3

If Q1 interacts with KCNE

Question 4

Study - Overexpression of KCNE1 or KCNQ1

Answer 4

Two-electrode voltage clamp Xenopus oocytes expressing cRNA encoding KCNQ1 & KCNQ1 + KCNE1
Q1+ E1 over exp:
Currents larger, slower activation , change in time in voltage dependence

Question 5

KCNE1 Knockout mice and Renal function

Answer 5

Looking at expression pattern using immunostaining in the Proximal tubule
- Majority of E1 expression in the proximal tubules and on the apical membrane
Suggests > E1 is regulating an apical membrane potassium channel likely to be Q1 channel
2nd paper shows-
However expression patterns don’t fit perfectly
A lot of Q1 in distal part of the tubule (of the nephron) some but not huge overlap

Question 6

Clearance studies in vivo - KO E1 or Q1 to look at kidney function
protocol

Answer 6

• Anaesthetise the mouse – look at depths by reflex test
• Cannulate jugular vein – fluid replacement
• Cannulate bladder- urine analysis look at diff conc of ions and solutes and vol per unit time
• Cannulate carodit- BP and Blood sample
• Heated pad to maintain body temp

Question 7

Clearance studies in vivo - KO E1 or Q1 to look at kidney function

Answer 7

Paper- E1 KO mice
Plasma Na and CL not sig different between two animals
Glomerular filtration rate – no sig difference
Plasma glucose lower in KO than WT but higher than normal but in both– should be 5-10 mM
GFR is not effected by KO of E1

Question 8

Human plasma glucose level

Answer 8

5-10 millimolar (mM)

Question 9

Fractional excretion

Answer 9

how much secreted per unit time divided by how much is filtered per unit time

Question 10

Clearance studies in vivo - KO E1 or Q1 to look at kidney function
looking at Fractional Excretion

Answer 10

Only small percentage being excreted
Struggling to reabsorb chloride
E1 KO losing sodium/excreting NA
Higher fractional excretion of glucose
Increase FE of fluid – increase urine flow rate
» Losing E1 is impacting the nephron somewhere

Question 11

FE of 100%

Answer 11

Everything thats being filtered is being secreted

Question 12

Sheffield Research

Answer 12

Initially tried to reproduce what was seen in the initial study

• looking at FE
• Increase FE of NA and CL
• No increase in glucose – no issues with reabsorption
• Would suggest problem later part of the proximal tubule
• Plasma Glucose about 10 mM – normal
• Increase in FE of water
• E1 important in the late proximal tubule - probably regulating K channel so has a role in regulating membrane potential

Question 13

Changes in FE

Answer 13

indication of changes in tubular function

Question 14

Is E1 regulating Q1?

Answer 14

Infuse chromanol 293b in the fluid – inhibitor of KCNQ1 in vivo studies

• Chromanol turns WT > E1 KO
• Chromanol no effect on KO
• E1 missing Q1 channels not working so nothing to block so no effect

Question 15

Is E1 regulating Q1?

Answer 15

Infuse chromanol 293b in the fluid – inhibitor of KCNQ1 in vivo studies

• Chromanol turns WT > mimics E1 KO
• Chromanol no effect on KO no change in chloride handling
• E1 missing Q1 channels not working so nothing to block so no effect
• • channel E1 is regulating has to be chromanol sensitive

Question 16

2. Given location data

Answer 16

probably PT, might also have a role more distally

Question 17

3. Lack of effect on glucose suggest

Answer 17

late PT (little glucose uptake

Question 18

Maintains -

Answer 18

Membrane potential and therefore transport function

Question 19

The Q1 inhibitor chromanol only has an effect in WT animals

Answer 19

Therefore KCNE1 regulating a chromanol sensitive K+ channel

So is it Q1?

Question 20

Study - Q1 KO

Answer 20

Clearance study
FE
measure Na and H20
under normal conditons dont see something that mimic E1
Main job of E1 doesn’t seem to be regulating Q1 K channels in the kidney

Question 21

Follow up - Patch Clamp technique

Answer 21

added chromanol and measured how much the current went down

• decrease in current is the chromanol sensitive component allows look at the C sensitive K channels
• look at graph
• should have blue shape - black and blue not the same
• *Suggests E1 regulating K channels that’s not Q1

Question 22

KCNE1 -Q1 summary overall conclusion

Answer 22

KCNE1 regulating another K channel that is not Q1.

There is some evidence that Q1 might play a small role

Question 23

KCNE2 & KCNQ1 - Gastric Function - Acid secretion in parietal cells

Answer 23

-Apical Cl channel allows secretion
-Chloride gradient established by a chloride bicarbonate exchanger
- Halogen ion secreted through proton/K atpase
cell secretes K across the membrane so that halogen ions can get in

Question 24

Stimulants

Answer 24

Ach- stimulates acid secretion through M3 ( muscarinic receptors)
Histamine- H2
Gastrin- CCKb

Question 25

Study - KCNQ1 KO & Gastric Function

- looking at ability of stomach cells secrete H+

Answer 25

Ammonium Pulse technique- Ammonia goes into the cell combines with H to be NH4+ - alkalisation

• remove outside ammonia so now driving force breaks apart the ammonium releases H ions really quickly
• acidification
• monitor over time - Ph recovers- recovery is a measure of how quickly hydrogen ions are being secreted by the cell
• in the absence of Na looking at the Proton/Potassium channel function - take away any contamination of the NA/H exchanger
• stimulate with Carbachol or histamine to stimulate H+ secretion

Question 26

Study - KCNQ1 KO & Gastric Function
- looking at ability of stomach cells secrete H+
PROTOCOL

Answer 26

Ammonium Pulse technique- Ammonia goes into the cell combines with H to be NH4+ - alkalisation

• remove outside ammonia so now driving force breaks apart the ammonium releases H ions really quickly
• acidification
• monitor over time - Ph recovers- recovery is a measure of how quickly hydrogen ions are being secreted by the cell
• in the absence of Na looking at the Proton/Potassium channel function - take away any contamination of the NA/H exchanger
• stimulate with Carbachol or histamine to stimulate H+ secretion

Question 27

Study - KCNQ1 KO & Gastric Function
- looking at ability of stomach cells secrete H+
RESULTS

Answer 27

WT - acidify then recover Ph quick
Q1 KO - no pH recovery because K channel isnt working so no K to come back into cell in exchange with H+ ions
- can inhibit acid secretion by just taking away the K+ channels

Question 28

KCNE2 KO & Gastric function-

Answer 28

KO- Achlorhydric (Less HCL in stomach)
Even though higher circulating gastrin levels (Gastrin increased to increase acid secretion)
compensation for low acidity in the stomach
-WT acidification with histamine
- +/- function relatively well still got half of E2
- KO - pH more alkaline so struggling to secrete acid

Question 29

KCNE2 KO & Gastric function- Ammonium Pulse Technique

Answer 29

Histamine stimulated
Parietal cells KO secrete less H+
-Red line E2 KO animals 
-Grey line from Hets 
-Black dotted line from Wt -absence of E2 no recovery Proton Potassium Atpase cant work no K secretion
E2 subunit not there to regulate Q1 
Reduction of H+ secretion 
KO Q1 or E2 see the same thing

Question 30

Acid Secretion model

Answer 30

K Channel on Apical side - KCNQ1/KCNE2

- Look at model