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BMS346 - Epithelial Physiology > L9 - Beta Subunits > Flashcards

L9 - Beta Subunits Flashcards

1
Q

What are beta subunits

A

Small proteins that may be cytosolic or integral membrane proteins
They interact with ion channels

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2
Q

How do beta subunits interact with ion channels

A

Through impacting on their trafficking and open prob

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3
Q

What is the KCNQ1 channel regulated by

A

KCNE

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4
Q

KCNQ1 is the

A

Alpha subunit

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5
Q

Normal function of an alpha subunit is only seen when

A

The beta subunit is alo present

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6
Q

How many members of the KCNE family

A

KCNE1-5

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7
Q

How many amnio acids in the E family

A

103-177

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8
Q

How many TMDs does the E family have

A

1 transmembrane domain

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9
Q

Describe the expression of KNCNE1-5

A

Expressed widely in excitable cells - implicated in long QT syndrome

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10
Q

What do the properties of Q1 depend on

A

The beta subunits that are regulating it

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11
Q

To investigate the effects of E1 on Q1 what experiements were performed

A

Two electrode vltage clamp on xeonopus oocytes expressing cRNA encoding Q1 and Q1andE1

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12
Q

Describe the differences in current seen when Q1 was expressed alone to when Q1 was expressed with E1

A

E1 enhances function of Q1

See a shift in time dependence - currents reach a steady state earlier when E1 present

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13
Q

Where is KCNE1 expreesed

A

In the proximal tubule

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14
Q

Describe the localisation of KCNE1

How does this relate to KNCQ1 expression

A

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-

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15
Q

What is the function of the proximal tubule

A

Early-mid important for the reabsorpition of glucose

Creates a driving force for Na uptake (also for glucose through the Na/glucose transporter

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16
Q

Describe the codnitoins for a clearance study

A

Anesthtised mouse on heated pad

Cannulate; carotid artery, jugular vein and bladder

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17
Q

Describe why the mouse is on a heated pad

A

To maintain a normal body temperature

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18
Q

Describe why the carotid pad needs to be cannulated

A

Ensure there is a viable BP
Can use this to determine the depth of anesthesia
Can take a blood sample at the end

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19
Q

Why is the jugular vein cannulated

A

To replace fluid

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20
Q

Why is the bladder cannulated

A

To measure volume per unit time - composition of the urine

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21
Q

Describe the plasma Na/Cl in the WT and E1 KO

A

Unchanged

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22
Q

Describe the plasma glucose levels in the WT vs E1 KO

Why might this not be accurate?

A

Plasma glucose is higher in the WT than E1 KO

Initially plasma glucose much higher than it should have been

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23
Q

What was seen RE GFR in the WT and E1 KO

A

GFR is unchanged in the wildtype and E1 KO

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24
Q

What does the term - fractional excretion of Na mean?

A

How much Na secreted per unit time as a fraction of the total ammount of Na that was filtered

25
Q

Describe the fractional excretion of Na, Cl, Glucose and fluid when performed in the original study

A

KO - higher Fe of Na, Cl, GLucose and fluid

26
Q

Describe the fraction excretion of Na, Cl, Glucose and H2O after it was redone - on animals with a normal plasma glucose

A

Fail to see any difference in the fractional excretion of glucsoe

Still see the increase Fe of Na Cl and fluid

27
Q

Why dont we expect to seee any effect of E1 on glucoseFe

A

Since E1 expressed in the late proximal tubule and most of the glucose has already been reabsorbed by this point

28
Q

What was used to investigate if E1 regulates Q1

A

Infuse a blocker of the Q1 channels

29
Q

What was the Q1 blocker used

A

Chromanol - 293b

30
Q

The application of chromonal mimics what

31
Q

What is the effect of chromonal on Fe of Na in the WT and the Q1 KO

A

Chromanol increase Fe of Na in WT

In the KO there is no effect – since already is a lack of E1

32
Q

What is the effect of chromonal on Fe of Cl in the WT and the Q1 KO

A

Chromanol increase Fe of Cl

In the KO there is no effect – since already is a lack of E1

33
Q

What is the effect of chromonal on Fe of fluid in the WT and the Q1 KO

A

Chromanol increases Fe of fluid

In the KO there is no effect – since already is a lack of E1

34
Q

KCNE1 is important in the regulation of what handling

A

Na, Cl, HCO3-

Water handling

35
Q

Q1 inhibitor only has an effect in

A

WT animals

36
Q

What do the results so far siggest

A

E1 is regulating a chromanol sensntivity channel - could this be Q1

37
Q

What do we expect to see in Q1 clearance studies

A

Expect phenotype to be the same as the E1 KO

38
Q

What do we actually see in the Q1 clearance studies

Conclusion

A

No change in Fe and Na and water in the Q1 KO

So its unlikely that E1 is regulating Q1

39
Q

What was measured in the patch clamp studies

A

Chromanol sensnitive currents- gives an idea of the function of the K channel

40
Q

Why was pA/pF used

A

To normalise the current to the size of the cell

41
Q

Describe the results of the patch clamp

Q1+E1 WT

E1 KO

Chromanol sensntive current

A

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

42
Q

Conclusions of the patch clamping experiments

A

KCNW1 regulates another channel that is not Q1

Some evidecne that Q1 might play a small role

43
Q

Gastric cell model

APICAL

A

APICAL

Chloride channel, K channel, K/H ATPase

44
Q

Gastric cell model

BASOLATERAL

A

Na/H exchnager
Na/K ATPase
K channel
HCO3-/Cl exchanger

45
Q

What are the two parts to gastric acid secretion

A

Cl secretion

H secretion

46
Q

Describe the Cl secretion part of gastric acid secretion

A

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

47
Q

Describe the H secretion pat of gastric acid secretion

A

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

48
Q

What three ways can acid secretion be stimulated

Which parts of the pathway does each one work on

A

ACh - M3
Histamine -H2
Gastrin - CCKb

49
Q

Describe the ammonium pulse technique

A

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

50
Q

Why was the ammonium pulse technique conducted in the absence of Na

A

To look at H secreting which have no requirement of Na - SO THE H/K ATPase

51
Q

Describe what was obsevred in the ammonium pulse technique for Q1 KO cells

A

No recovery

Until Na added the Na/H exchanger is now able to work so that recovers pH

52
Q

What is the reason for pariteal cells KO for Q1 secreting less H

A

Problem with apical K secretion

53
Q

What is the effect on stomach pH on histamine addition

A

Acid secretion

54
Q

What beta subunit was implicated in gastric acid secretion

55
Q

What is the effect of stomach pH in KCE2 KO

A

Response to histamine intact

56
Q

What is the effect of histamine on stomach pH in E2 KO cells

What is the compensation

A

No effect

Gastric levels are much higher as compensating for lack of acid secretion

57
Q

Describe the ammonium pulse technique results KO for E2

A

Parietal cells KO for E2 secreted less H as Q1 no longer functioning

58
Q

Describe what K channel mediates K secretion in gastric cells

A

K secretion mediated by KCNQ1 with the beta subunit KCNE2

BMS346 - Epithelial Physiology (10 decks)

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