L9 - Beta Subunits Flashcards
What kind of proteins are alpha subunits?
E.g. KCNQ1 – regulated by KCNE1 family
Integral membrane proteins
What kind of proteins are beta subunits?
Small molecular weight cytoplasmic/integral membrane proteins
Interact with ion channels – impact trafficking and function
Can be interconnected with alpha subunit
What does Barttin regulate?
CLCK Cl channel
How many members of the KCNE family are there?
KCNE1-5
What is the structure of the KCNE family?
Vary in size - 103 to 177 amino acids
1 transmembrane spanning domain
Where is the KCNE family expressed?
Widley expressed in excitable tissues – E family implicated in LQTS
When KCNQ1 mutated –> LQTS
Where are E1, E2 and E3 found?
Epithelial cells
Properties of KCNQ1 are determined by?
Which E1 subunit is regulating it
What happens to Q1 currents in the presence of E1?
Q1 currents larger
- E1 enhances function of Q1 channel
Shift in time dependence - currents reach steady state later
Where is E1 expressed?
E1 found on apical membrane of proximal tubule cells
What is the role of the early-mid proximal tubule?
Role in reabsorption of glucose
K channels in either apical or basolateral membrane together with Na/K ATPase set driving force (negative membrane potential) for Na uptake
Driving force for Na uptake and glucose comes with it
- Reabsorption from tubule fluid back into systemic circulation
Is there a link between the location of Q1 and E1 expression?
Q1 not found in exact same cell populations E1 is found
In clearance studies when mice are anaesthetised why do you measure temperature?
Rectal thermometer
If core body temp drops – signal is sent to heat up heating pad
In clearance studies when mice are anaesthetised why do you cannulate the jugular vein?
Fluid replacement
Saline and any test compounds
In clearance studies when mice are anaesthetised why do you cannulate the bladder?
Collect urine for analysis
Collect volume per unit time
In clearance studies when mice are anaesthetised why do you cannulate the carotid artery?
BP measurement and blood sample
BP used to determine depth of anaesthesia
Pinch test – if reflex response or BP spoke - need more anaesthesia
Do plasma Na and K concentrations differ between E1 KO and WT?
No
Do plasma glucose concentrations differ between E1 KO and WT?
Yes - lower in KO
Does GFR differ between E1 KO and WT?
No
Changes in urine contents are due to change in kidney function
What is fractional excretion?
How much Na excreted / how much Na filtered
If 50% - half of what Na is filtered is excreted
Does fractional excretion of Na, K or glucose differ between E1 KO and WT?
Yes - higher in KO
- Fluid higher as it follows Na
Indicates defects in function of mid-early proximal tubule – less driving force for Na reabsorption
- Must be mid to early as this is where glucose is absorbed
Overall what does the E1 KO do?
impacts ability of proximal tubule to reabsorb Na and Cl
Late proximal tubule doesn’t tend to absorb glucose
- Suggests E1 is found in the late proximal tubule
- If E1 is gone K channels not working – driving force for Na uptake is reduced
How did the test if E1 actually regulates Q1?
Used chromanol 293b - an inhibitor of KCNQ1
Infused into saline being injected in vivo – filtered by kidney
- If chromanol sensitive K channel on apical membrane where E1 is, the chromanol will block it so we will mimic KO E1 is wildtype animal
Is KCNQ1 chromanol sensitive?
Yes
What happened when they infused chromanol into the WT?
It turns it into a KO
What happened when they infused chromanol into the E1 KO?
Chromanol has no effect
Lack of E1 means K channels are not working anyway
Fractional excretion of Na and Cl already high in KO
Overall what did the experiments with chromonal show?
E1 is regulating chromanol 293b sensitive K channels
Sets driving force for Na, Cl and water reabsorption
What does the location data with E1 show?
KCNE1 found in proximal tubule - might also have a role more distally
- Lack of effect on glucose suggest late proximal tubule
- Little glucose uptake occurs in late proximal tubule
How did they test if the chromanol sensitive channel E1 was regulating was Q1?
Q1 KO studies
What were the results of Q1 KO studies?
Expected phenotype to be the same as E1 KO – not the case
Fractional excretion of Na and water same in WT and KO
- E1 in proximal tubule unlikely to be regulating Q1
Some differences under glucose loading
How did they use a patch clamp technique to show it was not Q1 being regulated?
Recorded chromanol sensitive currents in WT
Got currents – not mediated by Q1 (chromanol would have blocked this) –> E1 must be regulating a different K channel
In E1 knockout there is no current at all
What channels are found on the apical membrane of parietal cells in the stomach?
K channels
Cl channels
H+/K+ ATPase
What channels are found on the basolateral membrane of parietal cells in the stomach?
Na/K ATPase
K channel
Cl/HC03 exchanger
Na/halogen exchanger
What are the steps leading to net HCl secretion from parietal cells?
- Carbon dioxide and water diffuse into the cell
- Under influence of carbonic anhydrase –> production of bicarbonate and H ions
- Bicarbonate exchanges for Cl across basolateral membrane
- Increased intracellular concentration of Cl
- If we open Cl ion in apical membrane –> excretion of Cl
- H+/K+ ATPase uses ATP to exchange K coming into the cell for H leaving –> excretion of H
- Net secretion of HCl
What does H+/K+ ATPase rely on to allow H secretion?
K
Apical K channel – movement of K out of the cell is needed to drive H ion secretion
Important to maintain acid secretion
What are 3 stimulants of HCl secretion?
Ach –> muscarinic 3 receptors
Histamine –> H2 receptors
Gastrin –> CCKB receptors
Levels of these go up if the body detects there is insufficient acid in stomach
What is the ammonium pulse technique?
NH4+ in solution –> dissociated into NH3 and H+
NH3 moves into cell
NH3 mopes up H+ inside cells –> pH inside cell rises Take away NH4+ from outside cells
NH3 dumps H ions and leaves the cell –> pH inside cell drops –> acid load cells
NH3 moves in the opposite direction
Then look at rate of H+ excretion/pH recovery
What is the importance of doing the ammonium pulse technique in the absence of Na in the extracellular solution?
Then looking at H secreting pathways that don’t require Na
- Measurement of H+/K+ ATPase function
What is the importance of stimulating cells with carbachol or histamine?
Encourages H secretion
What happened to H excretion in Q1 KO?
H+/K+ ATPase function gone –> parietal cells secrete less H+
Q1 on apical membrane cannot excrete K+ –> no K for H+/K+ ATPase to function
Add Na –> Na/K+ ATPase now working –> pH recovers
What happens when you add histamine to wild type E2 cells?
Addition of histamine leads to acidification of stomach
What happens when you add histamine to KO E2 cells?
Addition of histamine does nothing
- Would normally upregulate Q1 and H+/K+ ATPase
- If Q1 not working as E2 not there –> no K secretion –> cant secrete HCl
Parietal cells secrete less H+ even though higher circulating gastrin level
- Mice know their acid stomach levels are not high enough
What is the pH like in KO E2 mice?
Higher than normal
What did the ammonium pulse technique with E2 KO show?
Lack of function of K/H ATPase
Because of a lack of function of KCNQ1 as its regulator E2 has been KO
pH slow to recover
A patient has a mutation in KCNE3. What is the impact of this mutation in terms of their upper airway cells and what symptoms might be observed?
KCNE3
- Basolateral membrane of upper airways
- It regulates KNCQ1 channel
LOF mutation means
- Q1 does not function normally
- Depolarisation of basolateral membrane potential
- Reduced driving force for NKCC1
- Less intracellular Cl
- Reduced driving force for Cl secretion
- Less Cl secretion
- Mimics CF – increased risk of infection, lung damage, early death
