Beta subunits Flashcards
What are beta subunits?
Cytoplasmic or integral membrane proteins that are able to modify the function, trafficking or regulation of the alpha subunit.
Which KCNE family members are found in epithelia and what characteristics do they have
KCNE1, 2 and 3
They range from 103 to 177 amino acids
They have 1 TMD
They are expressed in excitable cells
Regulate the KCNQ1 K channel
Do different members of the KCNE1 family have different effects on KCNLQT1? True or False
True - KCNE1 mutants can cause a complete loss in KCNQ1 function.
What is the effect of KCNE1 on KCNQ1 in oocytes expressing KCNQ1 and KCNQ1 with KCNE1
If you overexpress KCNE1 with Q1 enhances Q1 function (dont need to explain why)
Bigger currents as well as changing in gating.
What is the cell model for the proximal tubule.
Na/K ATPase on the basolateral side as well as a K channel as well as a glucose channel
On the apical membrane there is a Na/Glucose co-transporter which utilises the sodium gradient created to transport Na across with glucose which is then reabsorbed across the basolateral down its conc grad.
This also drives the reabsorption of chloride in the paracellular pathway as well as water.
Where is KCNE1 protein expressed in the kidney?
Blue stain is the nuclei, Green is KCNE1
Rings of green staining is the apical membrane (brush border) - Suggests there is a K channel in the apical channel which is helping set up the driving force for Na reabsorption.
How does KCNE1 and KCNQ1 expression differ?
Again expressed in the apical membrane of proximal tubule cells
But not in all the same cells as Q1
Therefore there are cells with KCNQ1 alone, some with KCNE1 with Q1 and some with KCNE1 alone (it will be modulating the function of a different K channel)
What was the in vivo clearence study used to investigate KCNE1
Mice are placed on a heated pad after being anaesthetised, whilst anaesthetised mice struggle to regulate their body tempreature (hence the heated pad)
The carotid artery is canulated for BP measurements and blood sampling (at the end)
Cannulate the jugular vein to replace fluids lost.
Cannulate the bladder of the animals and collect the urine for analysis.
What is the effect of a KCNE1 KO on plasma Na and Cl concentration
Nothing
What is the effect of a KO of KCNE1 on glucose concentrations in the plasma.
Plamsa glucose levels are the same
one study said there was a reduction but they had stupidly high glucose concentrations (probably was just the animal attempting to clear the extra glucose)
What is the effect of a KCNE1 KO on GFR
Nothing
What is the effect of KCNE1 KO on fractional excretion (rate of excretion/rate of filtration) x100 - (100% means everything filtered is being excreted)
What were the effects on Na, Cl, Glucose and fluid
Na excretion is around 3 times larger - problem reabsorbing sodium somewhere along the nephron
Cl is also around 2 times larger - “ “
Also an increased fractional excretion of glucose - explains the reduction of plasma glucose seen in these animals)
Also an increase in urine flow rate - makes sense given the sodium and chloride increased secretion.
KCNE1 regulates a K channel in the kidney plays a role in helping the kidney drive some Na, Cl and glucose reabsorption.
What effect does chromanol 293B have on Na, Cl and water handling in WT and KCNE1 KOs
Chromanol 293B in WT is blocking Q1 channels, hindering Na reabsorption and therefore increased Na loss in urine.
Chromanol 293B has no sig diff on the KCNE1 KO. E1 regulates Q1 so if its missing there is no Q1 action.
Same profile for Cl and water handling.
Does the chromanol 293B data prove that E1 is regulating KCNQ1
Not for sure, as other channels are chromanol 293B sensitive. But it is a channel that is 293B sensitive, and is regulated by KCNE1
What summary was made after the in vivo studies from the first part of the lecture
KCNE1 is important in Na, Cl and HCO3 and therefore water handling
Given location data - Probably playing a role in the proximal tubule
The lack of effect on plasma glucose suggests its in the latter part of the PT as this is where there is very little glucose handling (so lack of KCNE1 would have little effect)
Maintains membrane potential and therefore transport function
What would we expect of the phenotype of a KCNQ1 KO mouse
Expect it to be the same as KCNE1 KO as E1 is regulating Q1 so should have a similar effect. i.e an increase in FE of Cl, Na and water
Do KCNQ1 KO mice have the same phenotype as KCNE1 KO mice.
No
No significant difference in FE of Na or water under normal conditions
What is the the effect of knocking out KCNE1 on chromanol 293B sensitive current in PT cells.
What can be taken from the study
Black is the WT PT cells. These are very K sensitive
Red is the E1 KO - There is no current, so the channels that E1 is regulating require its function.
The black IV curve looks nothing like the KCNQ1/E1 complex (blue line) - suggests the K channel regulated by E1 is NOT KCNQ1
What are the stimulants of acid secretion of the parietal cells in the stomach. (and what receptors)
Ach - M3 receptors
Histamine - H2 receptors
Gastrin - CCKb receptors
All on the basolateral membrane
What is the model for the parietal cell acid secretion secretion
Carbon dioxise and water go into the cells. Water dissociates to form hydrogen ions and hydroxide ions. Hydroxide ions combine with carbon dioxide to from bicarbonate. Bicarbonate recycles across the basolateral membrane in exchange for chloride. Chloride accumulates inside the cell and will eventually be secreted at the apical membrane by a chloride channel.
What is the importance of the apical K channel in parital cells of the stomach
It secretes K into the lumen of the stomach, these ions then get recycled through the K/H ATPase. This allows for the secretion of H ions into the lumen of the stomach. Without this K channel there is limited H ion secretion into the lumen
What is the ammonium pulse technique
Expose the cells to ammonium. Some is dissociated into NH3 and H. NH3 freely diffuses into the cell and then combines with H ions to make the intracellular pH more alkali.
When you take away the extracellular solution there is a reversal in the driving force. NH4 again dissociates and moves back out the cell, creating a large acidification (as more H ions are left inside the cell then previously). This acidification causes an upregulation of acid secretion mechanisms
If you do an analysis of how quickly the cell recoveres from the acidification it is a measure of the function of the acid secreting mechanisms.
If you do this without any Na around then the action of the Na/H exchanger is removed and in the case of the parietal cells the recovery phase is a measurement of the function of the H/K ATPase.
What is the effect of a KCNQ1 KO on the recovery phase of the parietal cells.
There is no recovery. Not enough potassium secretion from the apical membrane so not enough potassium for the action of the K/H ATPase action and acid secretion.
If you then add Na back in then there is a recovery due to the Na/H exchanger on the basolateral membrane.
What is the effect of a KCNE2 KO on stomach pH (after histamine stimulation)
reduction in pH after stimulation of parietal cells in WT
In KCNE2 KO homozygotes, the baseling pH is more alkali and there is no response to histamine.
How are KCNE2 mice described and what does this mean
Achlorhydric - they are struggling to secrete acid by the parietal cells
They have higher circulating gastrin levels which is a compensatory mechanism. Gastrin is a hormone that stimulates acid secretion.
What is the effect of KCNE2 KO on the ammonium pulse chase experiment
Following the removal of the extracellular solution there is no recovery in the KO animals compared to WT. Very similar shape to the KCNQ1 KO pulse chase.
