L9 - Beta Subunits Flashcards
What are beta subunits
Small proteins that may be cytosolic or integral membrane proteins
They interact with ion channels
How do beta subunits interact with ion channels
Through impacting on their trafficking and open prob
What is the KCNQ1 channel regulated by
KCNE
KCNQ1 is the
Alpha subunit
Normal function of an alpha subunit is only seen when
The beta subunit is alo present
How many members of the KCNE family
KCNE1-5
How many amnio acids in the E family
103-177
How many TMDs does the E family have
1 transmembrane domain
Describe the expression of KNCNE1-5
Expressed widely in excitable cells - implicated in long QT syndrome
What do the properties of Q1 depend on
The beta subunits that are regulating it
To investigate the effects of E1 on Q1 what experiements were performed
Two electrode vltage clamp on xeonopus oocytes expressing cRNA encoding Q1 and Q1andE1
Describe the differences in current seen when Q1 was expressed alone to when Q1 was expressed with E1
E1 enhances function of Q1
See a shift in time dependence - currents reach a steady state earlier when E1 present
Where is KCNE1 expreesed
In the proximal tubule
Describe the localisation of KCNE1
How does this relate to KNCQ1 expression
In rings - apical membrane of proximal tubule cells of the mouse
Some overlap in E1/Q1 but also many areas that are E1+ and Q1-
What is the function of the proximal tubule
Early-mid important for the reabsorpition of glucose
Creates a driving force for Na uptake (also for glucose through the Na/glucose transporter
Describe the codnitoins for a clearance study
Anesthtised mouse on heated pad
Cannulate; carotid artery, jugular vein and bladder
Describe why the mouse is on a heated pad
To maintain a normal body temperature
Describe why the carotid pad needs to be cannulated
Ensure there is a viable BP
Can use this to determine the depth of anesthesia
Can take a blood sample at the end
Why is the jugular vein cannulated
To replace fluid
Why is the bladder cannulated
To measure volume per unit time - composition of the urine
Describe the plasma Na/Cl in the WT and E1 KO
Unchanged
Describe the plasma glucose levels in the WT vs E1 KO
Why might this not be accurate?
Plasma glucose is higher in the WT than E1 KO
Initially plasma glucose much higher than it should have been
What was seen RE GFR in the WT and E1 KO
GFR is unchanged in the wildtype and E1 KO
What does the term - fractional excretion of Na mean?
How much Na secreted per unit time as a fraction of the total ammount of Na that was filtered
Describe the fractional excretion of Na, Cl, Glucose and fluid when performed in the original study
KO - higher Fe of Na, Cl, GLucose and fluid
Describe the fraction excretion of Na, Cl, Glucose and H2O after it was redone - on animals with a normal plasma glucose
Fail to see any difference in the fractional excretion of glucsoe
Still see the increase Fe of Na Cl and fluid
Why dont we expect to seee any effect of E1 on glucoseFe
Since E1 expressed in the late proximal tubule and most of the glucose has already been reabsorbed by this point
What was used to investigate if E1 regulates Q1
Infuse a blocker of the Q1 channels
What was the Q1 blocker used
Chromanol - 293b
The application of chromonal mimics what
Q1 KO
What is the effect of chromonal on Fe of Na in the WT and the Q1 KO
Chromanol increase Fe of Na in WT
In the KO there is no effect – since already is a lack of E1
What is the effect of chromonal on Fe of Cl in the WT and the Q1 KO
Chromanol increase Fe of Cl
In the KO there is no effect – since already is a lack of E1
What is the effect of chromonal on Fe of fluid in the WT and the Q1 KO
Chromanol increases Fe of fluid
In the KO there is no effect – since already is a lack of E1
KCNE1 is important in the regulation of what handling
Na, Cl, HCO3-
Water handling
Q1 inhibitor only has an effect in
WT animals
What do the results so far siggest
E1 is regulating a chromanol sensntivity channel - could this be Q1
What do we expect to see in Q1 clearance studies
Expect phenotype to be the same as the E1 KO
What do we actually see in the Q1 clearance studies
Conclusion
No change in Fe and Na and water in the Q1 KO
So its unlikely that E1 is regulating Q1
What was measured in the patch clamp studies
Chromanol sensnitive currents- gives an idea of the function of the K channel
Why was pA/pF used
To normalise the current to the size of the cell
Describe the results of the patch clamp
Q1+E1 WT
E1 KO
Chromanol sensntive current
WT - normal opening of the channels
E1 KO - no function of the votlage senstive channels without the beta subunit
Chromanol sensitive - Dont show any voltage dependence - BUT THERE ARE CURRENTS PRESENT
Conclusions of the patch clamping experiments
KCNW1 regulates another channel that is not Q1
Some evidecne that Q1 might play a small role
Gastric cell model
APICAL
APICAL
Chloride channel, K channel, K/H ATPase
Gastric cell model
BASOLATERAL
Na/H exchnager
Na/K ATPase
K channel
HCO3-/Cl exchanger
What are the two parts to gastric acid secretion
Cl secretion
H secretion
Describe the Cl secretion part of gastric acid secretion
CO2 and H20 diffuse into the parietal cells
Under the influence of carbonic anhydrase they form bicarbonate and hydrogen ions
Biocarbonate leaves through the Cl/HCO3 exchanger (Cl moves in)
This increases the concentration of IC Cl leading to a driving force for Cl secretion
Cl out through apical Cl channels - NET SECRETION OF CL
Describe the H secretion pat of gastric acid secretion
H/K ATPase on the apical membrane has an absolute requirement for K
Movement of K out of the cell through apical K channels is required to support the next secretion of H
What three ways can acid secretion be stimulated
Which parts of the pathway does each one work on
ACh - M3
Histamine -H2
Gastrin - CCKb
Describe the ammonium pulse technique
NH4 applie and dissociates - NH3 moves into the cell and mops up H ions on the inside - increase in pH
Then REMOVE THE AMMONIUM NH3 moves out dumping its H ions - large acidication - decrease in IC pH
CAN THEN MEASURE THE RATE OF PH RECOVERY
Why was the ammonium pulse technique conducted in the absence of Na
To look at H secreting which have no requirement of Na - SO THE H/K ATPase
Describe what was obsevred in the ammonium pulse technique for Q1 KO cells
No recovery
Until Na added the Na/H exchanger is now able to work so that recovers pH
What is the reason for pariteal cells KO for Q1 secreting less H
Problem with apical K secretion
What is the effect on stomach pH on histamine addition
Acid secretion
What beta subunit was implicated in gastric acid secretion
KCNE2
What is the effect of stomach pH in KCE2 KO
Response to histamine intact
What is the effect of histamine on stomach pH in E2 KO cells
What is the compensation
No effect
Gastric levels are much higher as compensating for lack of acid secretion
Describe the ammonium pulse technique results KO for E2
Parietal cells KO for E2 secreted less H as Q1 no longer functioning
Describe what K channel mediates K secretion in gastric cells
K secretion mediated by KCNQ1 with the beta subunit KCNE2
