L9 - Beta Subunits

Question 1

What kind of proteins are alpha subunits?

Answer 1

E.g. KCNQ1 – regulated by KCNE1 family

Integral membrane proteins

Question 2

What kind of proteins are beta subunits?

Answer 2

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

Question 3

What does Barttin regulate?

Answer 3

CLCK Cl channel

Question 4

How many members of the KCNE family are there?

Answer 4

KCNE1-5

Question 5

What is the structure of the KCNE family?

Answer 5

Vary in size - 103 to 177 amino acids

1 transmembrane spanning domain

Question 6

Where is the KCNE family expressed?

Answer 6

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

Question 7

Where are E1, E2 and E3 found?

Answer 7

Epithelial cells

Question 8

Properties of KCNQ1 are determined by?

Answer 8

Which E1 subunit is regulating it

Question 9

What happens to Q1 currents in the presence of E1?

Answer 9

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

Question 10

Where is E1 expressed?

Answer 10

E1 found on apical membrane of proximal tubule cells

Question 11

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

Answer 11

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

Question 12

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

Answer 12

Q1 not found in exact same cell populations E1 is found

Question 13

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

Answer 13

Rectal thermometer

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

Question 14

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

Answer 14

Fluid replacement

Saline and any test compounds

Question 15

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

Answer 15

Collect urine for analysis

Collect volume per unit time

Question 16

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

Answer 16

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

Question 17

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

Answer 17

No

Question 18

Do plasma glucose concentrations differ between E1 KO and WT?

Answer 18

Yes - lower in KO

Question 19

Does GFR differ between E1 KO and WT?

Answer 19

No

Changes in urine contents are due to change in kidney function

Question 20

What is fractional excretion?

Answer 20

How much Na excreted / how much Na filtered

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

Question 21

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

Answer 21

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

Question 22

Overall what does the E1 KO do?

Answer 22

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

Question 23

How did the test if E1 actually regulates Q1?

Answer 23

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

Question 24

Is KCNQ1 chromanol sensitive?

Answer 24

Yes

Question 25

What happened when they infused chromanol into the WT?

Answer 25

It turns it into a KO

Question 26

What happened when they infused chromanol into the E1 KO?

Answer 26

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

Question 27

Overall what did the experiments with chromonal show?

Answer 27

E1 is regulating chromanol 293b sensitive K channels | Sets driving force for Na, Cl and water reabsorption

Question 28

What does the location data with E1 show?

Answer 28

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

Question 29

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

Answer 29

Q1 KO studies

Question 30

What were the results of Q1 KO studies?

Answer 30

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

Question 31

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

Answer 31

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

Question 32

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

Answer 32

K channels Cl channels H+/K+ ATPase

Question 33

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

Answer 33

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

Question 34

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

Answer 34

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

Question 35

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

Answer 35

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

Question 36

What are 3 stimulants of HCl secretion?

Answer 36

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

Question 37

What is the ammonium pulse technique?

Answer 37

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

Question 38

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

Answer 38

Then looking at H secreting pathways that don’t require Na | - Measurement of H+/K+ ATPase function

Question 39

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

Answer 39

Encourages H secretion

Question 40

What happened to H excretion in Q1 KO?

Answer 40

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

Question 41

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

Answer 41

Addition of histamine leads to acidification of stomach

Question 42

What happens when you add histamine to KO E2 cells?

Answer 42

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

Question 43

What is the pH like in KO E2 mice?

Answer 43

Higher than normal

Question 44

What did the ammonium pulse technique with E2 KO show?

Answer 44

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

Question 45

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?

Answer 45

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