Lecture 10 Beta Subunits

Question 1

What are alpha subunits?

Give an example

Answer 1

the integral membrane protein

KCNQ1

Question 2

What are beta subunits?

Answer 2

cytoplasmic/integral membrane proteins that act to modify the properties of the alpha subunit
KCNE family (1-5); Barttin

Question 3

What pathology is KCNQ1 linked to?

How do beta subunits affect this?

Answer 3

long QT syndrome
different subunits with varying numbers of amino acids and one transmembrane domain have different effects on the channel - KCNE1-3

Question 4

What affect does KCNE1 have on KCNQ1? What experimental evidence is there for this?

What is the importance of this?

Answer 4

two electrode voltage clamp on Xenopus oocytes expressing cRNA for Q1 alone or Q1 and E1 together
Q1 alone - current increases and potential increases
Q1 and E1 - gating of voltage dependence has shifted. lager currents

important physiologically in terms of organ function

Question 5

Where is the majority of E1 expressed?
Where is the majority of Q1 expressed?
What does this impact?
What experimental evidence is there for this?

Answer 5

apical membrane of proximal tubule cells of the kidney
apical membrane of some proximal tubule cells and distal tubule cells of the kidney
likely E1 regulating an apical membrane protein but expression profiles of E1 and Q1 not exactly the same
immunostaining of E1 and Q1

Question 6

How are clearance studies (in vivo) carried out on mice?

Answer 6

anaesthetise mouse and maintain physiological conditions:
cannulate carotid artery to measure blood pressure and blood sample for ion and solute levels - low BP indicates low perfusion to the kidney
cannulate jugular vein - fluid replacement increasing BP
animal on heated pad - maintain body temperature
cannulate bladder - collect urine for analysis

Question 7

What did E1 knock-out mice show from clearance studies?

What was the impact on fractional excretion on the E1 KO?

Answer 7

no effect on sodium and chloride plasma concentrations
no effect on GFR
significantly decreased plasma glucoside levels compared to normal BUT very high glucose levels compared to human standard values. Careful with data

increased sodium and chloride excretion
higher fluid excretion
these signal changes in the tubular function

Question 8

Define fractional excretion (FE):

Answer 8

(the rate of excretion / the rate of filtration ) x 100%

if FE is 100% everything being filtered is being excreted

Question 9

What is the problem with glucose excretion for the E1 KO?

Answer 9

some data shows high FE alongside abnormally high glucose levels in the plasma (<100mM when in humans is 4-6mM)
other data with normal glucose ranges show no change in [glucose] between wt and KO indicating the late proximal tubule is the main expression of E1

Question 10

From clearance studies what is the most likely role of E1 on Q1?

Answer 10

setting the membrane potential through controlling reabsorption of sodium, chloride and water

Question 11

What is the function of chromanol 293b?

How is it incorporated into the clearance studies?

Answer 11

inhibits Q1

infused at a high enough dose to impact the whole kidney but not effect the cardiac muscle (max dose)

Question 12

What effect does chromanol 293b have on Q1?
What effect does chromanol 293b have on E1 KO?

What does this suggest?

Answer 12

Q1 - reduced FE of sodium, chloride and water reabsorption
E1 KO - chromanol 293b has no effect on E1 KO
blocking Q1 gives a similar response to knocking out E1 however chromanol 293b only effects Q1

Question 13

How is the FE affected in Q1 KO mice?

Answer 13

no effect on sodium and water under normal conditions suggesting that Q1 and E1 are not related

Question 14

Does E1 regulate Q1 in the proximal tubule? What evidence is there for this?

Answer 14

no, E1 is regulating another potassium channel that is not Q1
whole cell patch clamp technique on dissected late proximal tubules with chromanol to measure the chromanol-sensitive component of the potassium channels
wild type current very different to the voltage-dependent curve
E1 KO showed no chromanol-sensitive current indicating that all chromanol-sensitive channels in the proximal tubule are regulated by E1

Question 15

Where else can Q1 be found?
What is it’s function there?
What beta-subunit regulates it?

Answer 15

found in stomach
gastric function and acid secretion in parietal cells - chloride-bicarbonate exchanger
E2 (KCNE2)

Question 16

What impact does KO of Q1 have on gastric function?

What experimental evidence is there for this?

Answer 16

Q1 KO in parietal cells lose the ability to effectively secrete acid to maintain a constant pH

ammonium pulse technique - remove Na+ to stop contamination from Na/K-ATPase in BL membrane
add NH3 to cells which forms NH4+ by binding with H+ and an alkalisation
remove the NH3 from the outside of the cells which break down NH4+ to release H+ and give acidification
look how quickly cell pH recovers
in KO pH cannot recover without sodium due to no potassium secretion across the apical membrane making the potassium-hydrogen exchanger unable to work

Question 17

Give an example of three acid secreting stimulants on parietal cells? What receptors do they interact with?

Answer 17

acetylcholine - muscarinic M3 receptors
histamine - H2 receptors
gastrin - CCK-beta receptors

Question 18

What is observed in the animals as a result of E2 KO?

Answer 18

achlorhydric condition where not enough acid (HCl) in the stomach despite high circulating levels of gastrin

Question 19

How do animals respond to histamine in terms of E2 and E2 KO?

Answer 19

in wild type of heterozygous animals for the E2 protein an acidification is observed in response to histamine
in homozygous (KO) animals for the E2 protein they have higher than normal pH of the stomach (reduced acid) as well as loss of response to histamine

Question 20

What happens in the stomach if you KO Q1?

Answer 20

no Q1 alpha subunit of the potassium channel 
reduced potassium secretion
reduced potassium-hydrogen ion exchange
decreased hydrogen ion secretion
increased pH of stomach