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Epithelial Physiology > L9 - Beta Subunits > Flashcards

L9 - Beta Subunits Flashcards

1
Q

What kind of proteins are alpha subunits?

A

E.g. KCNQ1 – regulated by KCNE1 family

Integral membrane proteins

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2
Q

What kind of proteins are beta subunits?

A

Small molecular weight cytoplasmic/integral membrane proteins
Interact with ion channels – impact trafficking and function
Can be interconnected with alpha subunit

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3
Q

What does Barttin regulate?

A

CLCK Cl channel

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4
Q

How many members of the KCNE family are there?

A

KCNE1-5

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5
Q

What is the structure of the KCNE family?

A

Vary in size - 103 to 177 amino acids

1 transmembrane spanning domain

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6
Q

Where is the KCNE family expressed?

A

Widley expressed in excitable tissues – E family implicated in LQTS
When KCNQ1 mutated –> LQTS

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7
Q

Where are E1, E2 and E3 found?

A

Epithelial cells

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8
Q

Properties of KCNQ1 are determined by?

A

Which E1 subunit is regulating it

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9
Q

What happens to Q1 currents in the presence of E1?

A

Q1 currents larger
- E1 enhances function of Q1 channel
Shift in time dependence - currents reach steady state later

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10
Q

Where is E1 expressed?

A

E1 found on apical membrane of proximal tubule cells

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11
Q

What is the role of the early-mid proximal tubule?

A

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

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12
Q

Is there a link between the location of Q1 and E1 expression?

A

Q1 not found in exact same cell populations E1 is found

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13
Q

In clearance studies when mice are anaesthetised why do you measure temperature?

A

Rectal thermometer

If core body temp drops – signal is sent to heat up heating pad

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14
Q

In clearance studies when mice are anaesthetised why do you cannulate the jugular vein?

A

Fluid replacement

Saline and any test compounds

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15
Q

In clearance studies when mice are anaesthetised why do you cannulate the bladder?

A

Collect urine for analysis

Collect volume per unit time

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16
Q

In clearance studies when mice are anaesthetised why do you cannulate the carotid artery?

A

BP measurement and blood sample
BP used to determine depth of anaesthesia
Pinch test – if reflex response or BP spoke - need more anaesthesia

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17
Q

Do plasma Na and K concentrations differ between E1 KO and WT?

A

No

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18
Q

Do plasma glucose concentrations differ between E1 KO and WT?

A

Yes - lower in KO

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19
Q

Does GFR differ between E1 KO and WT?

A

No

Changes in urine contents are due to change in kidney function

20
Q

What is fractional excretion?

A

How much Na excreted / how much Na filtered

If 50% - half of what Na is filtered is excreted

21
Q

Does fractional excretion of Na, K or glucose differ between E1 KO and WT?

A

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

22
Q

Overall what does the E1 KO do?

A

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

23
Q

How did the test if E1 actually regulates Q1?

A

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

24
Q

Is KCNQ1 chromanol sensitive?

A

Yes

25
Q

What happened when they infused chromanol into the WT?

A

It turns it into a KO

26
Q

What happened when they infused chromanol into the E1 KO?

A

Chromanol has no effect
Lack of E1 means K channels are not working anyway
Fractional excretion of Na and Cl already high in KO

27
Q

Overall what did the experiments with chromonal show?

A

E1 is regulating chromanol 293b sensitive K channels

Sets driving force for Na, Cl and water reabsorption

28
Q

What does the location data with E1 show?

A

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
29
Q

How did they test if the chromanol sensitive channel E1 was regulating was Q1?

A

Q1 KO studies

30
Q

What were the results of Q1 KO studies?

A

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

31
Q

How did they use a patch clamp technique to show it was not Q1 being regulated?

A

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

32
Q

What channels are found on the apical membrane of parietal cells in the stomach?

A

K channels
Cl channels
H+/K+ ATPase

33
Q

What channels are found on the basolateral membrane of parietal cells in the stomach?

A

Na/K ATPase
K channel
Cl/HC03 exchanger
Na/halogen exchanger

34
Q

What are the steps leading to net HCl secretion from parietal cells?

A
  1. Carbon dioxide and water diffuse into the cell
  2. Under influence of carbonic anhydrase –> production of bicarbonate and H ions
  3. Bicarbonate exchanges for Cl across basolateral membrane
  4. Increased intracellular concentration of Cl
  5. If we open Cl ion in apical membrane –> excretion of Cl
  6. H+/K+ ATPase uses ATP to exchange K coming into the cell for H leaving –> excretion of H
  7. Net secretion of HCl
35
Q

What does H+/K+ ATPase rely on to allow H secretion?

A

K
Apical K channel – movement of K out of the cell is needed to drive H ion secretion
Important to maintain acid secretion

36
Q

What are 3 stimulants of HCl secretion?

A

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

37
Q

What is the ammonium pulse technique?

A

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

38
Q

What is the importance of doing the ammonium pulse technique in the absence of Na in the extracellular solution?

A

Then looking at H secreting pathways that don’t require Na

- Measurement of H+/K+ ATPase function

39
Q

What is the importance of stimulating cells with carbachol or histamine?

A

Encourages H secretion

40
Q

What happened to H excretion in Q1 KO?

A

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

41
Q

What happens when you add histamine to wild type E2 cells?

A

Addition of histamine leads to acidification of stomach

42
Q

What happens when you add histamine to KO E2 cells?

A

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

43
Q

What is the pH like in KO E2 mice?

A

Higher than normal

44
Q

What did the ammonium pulse technique with E2 KO show?

A

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

45
Q

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?

A

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

Epithelial Physiology (10 decks)

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