L9 - Beta Subunits Flashcards by Jack Corston

What are beta subunits

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

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How do beta subunits interact with ion channels

Through impacting on their trafficking and open prob

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What is the KCNQ1 channel regulated by

KCNE

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KCNQ1 is the

Alpha subunit

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Normal function of an alpha subunit is only seen when

The beta subunit is alo present

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How many members of the KCNE family

KCNE1-5

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How many amnio acids in the E family

103-177

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How many TMDs does the E family have

1 transmembrane domain

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Describe the expression of KNCNE1-5

Expressed widely in excitable cells - implicated in long QT syndrome

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What do the properties of Q1 depend on

The beta subunits that are regulating it

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

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

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

E1 enhances function of Q1

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

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Where is KCNE1 expreesed

In the proximal tubule

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Describe the localisation of KCNE1

How does this relate to KNCQ1 expression

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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What is the function of the proximal tubule

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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Describe the codnitoins for a clearance study

Anesthtised mouse on heated pad

Cannulate; carotid artery, jugular vein and bladder

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Describe why the mouse is on a heated pad

To maintain a normal body temperature

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Describe why the carotid pad needs to be cannulated

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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Why is the jugular vein cannulated

To replace fluid

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Why is the bladder cannulated

To measure volume per unit time - composition of the urine

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

Unchanged

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Describe the plasma glucose levels in the WT vs E1 KO

Why might this not be accurate?

Plasma glucose is higher in the WT than E1 KO

Initially plasma glucose much higher than it should have been

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What was seen RE GFR in the WT and E1 KO

GFR is unchanged in the wildtype and E1 KO

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What does the term - fractional excretion of Na mean?

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

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

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

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

Fail to see any difference in the fractional excretion of glucsoe Still see the increase Fe of Na Cl and fluid

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

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

What was used to investigate if E1 regulates Q1

Infuse a blocker of the Q1 channels

What was the Q1 blocker used

Chromanol - 293b

The application of chromonal mimics what

Q1 KO

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

Chromanol increase Fe of Na in WT | In the KO there is no effect – since already is a lack of E1

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

Chromanol increase Fe of Cl | In the KO there is no effect – since already is a lack of E1

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

Chromanol increases Fe of fluid | In the KO there is no effect – since already is a lack of E1

KCNE1 is important in the regulation of what handling

Na, Cl, HCO3- | Water handling

Q1 inhibitor only has an effect in

WT animals

What do the results so far siggest

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

What do we expect to see in Q1 clearance studies

Expect phenotype to be the same as the E1 KO

What do we actually see in the Q1 clearance studies Conclusion

No change in Fe and Na and water in the Q1 KO So its unlikely that E1 is regulating Q1

What was measured in the patch clamp studies

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

Why was pA/pF used

To normalise the current to the size of the cell

Describe the results of the patch clamp Q1+E1 WT E1 KO Chromanol sensntive current

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

Conclusions of the patch clamping experiments

KCNW1 regulates another channel that is not Q1 | Some evidecne that Q1 might play a small role

Gastric cell model | APICAL

APICAL | Chloride channel, K channel, K/H ATPase

Gastric cell model | BASOLATERAL

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

What are the two parts to gastric acid secretion

Cl secretion | H secretion

Describe the Cl secretion part of gastric acid secretion

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

Describe the H secretion pat of gastric acid secretion

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

What three ways can acid secretion be stimulated Which parts of the pathway does each one work on

ACh - M3 Histamine -H2 Gastrin - CCKb

Describe the ammonium pulse technique

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

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

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

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

No recovery | Until Na added the Na/H exchanger is now able to work so that recovers pH

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

Problem with apical K secretion

What is the effect on stomach pH on histamine addition

Acid secretion

What beta subunit was implicated in gastric acid secretion

KCNE2

What is the effect of stomach pH in KCE2 KO

Response to histamine intact

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

No effect Gastric levels are much higher as compensating for lack of acid secretion

Describe the ammonium pulse technique results KO for E2

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

Describe what K channel mediates K secretion in gastric cells

K secretion mediated by KCNQ1 with the beta subunit KCNE2