Lecture 10 - Beta Subunit interactions Flashcards
• Define subunits, giving examples • Describe KCNE family • Discuss KCNE expression epithelia • Discuss the evidence that KCNE1 regulates K+ channels in kidney • Discuss the evidence that KCNE2 regulates K+ channels in stomach
Beta Subunits
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
KCNE Family
KCNE1- 103 to 177 amino acids
KCNE2 - 1 TM domain
KCNE3 - Excitable - Long QT syndrome
KCNQ1- Long QT syndrome
Varying effect on function
If Q1 interacts with KCNE
Study - Overexpression of KCNE1 or KCNQ1
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
KCNE1 Knockout mice and Renal function
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
Clearance studies in vivo - KO E1 or Q1 to look at kidney function
protocol
- 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
Clearance studies in vivo - KO E1 or Q1 to look at kidney function
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
Human plasma glucose level
5-10 millimolar (mM)
Fractional excretion
how much secreted per unit time divided by how much is filtered per unit time
Clearance studies in vivo - KO E1 or Q1 to look at kidney function
looking at Fractional Excretion
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
FE of 100%
Everything thats being filtered is being secreted
Sheffield Research
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
Changes in FE
indication of changes in tubular function
Is E1 regulating Q1?
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
Is E1 regulating Q1?
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
- Given location data
probably PT, might also have a role more distally
- Lack of effect on glucose suggest
late PT (little glucose uptake
Maintains -
Membrane potential and therefore transport function
The Q1 inhibitor chromanol only has an effect in WT animals
Therefore KCNE1 regulating a chromanol sensitive K+ channel
So is it Q1?
Study - Q1 KO
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
Follow up - Patch Clamp technique
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
KCNE1 -Q1 summary overall conclusion
KCNE1 regulating another K channel that is not Q1.
There is some evidence that Q1 might play a small role
KCNE2 & KCNQ1 - Gastric Function - Acid secretion in parietal cells
-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
Stimulants
Ach- stimulates acid secretion through M3 ( muscarinic receptors)
Histamine- H2
Gastrin- CCKb
Study - KCNQ1 KO & Gastric Function
- looking at ability of stomach cells secrete H+
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
Study - KCNQ1 KO & Gastric Function
- looking at ability of stomach cells secrete H+
PROTOCOL
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
Study - KCNQ1 KO & Gastric Function
- looking at ability of stomach cells secrete H+
RESULTS
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
KCNE2 KO & Gastric function-
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
KCNE2 KO & Gastric function- Ammonium Pulse Technique
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
Acid Secretion model
K Channel on Apical side - KCNQ1/KCNE2
- Look at model
