Lecture 8 Beta Subunits

Question 1

What attributes of the α subunit can be regulated by β or accessory subunits

Answer 1

Regulation trafficking expression and function

Question 2

Outline the different ways β subunits can associate with the channel

Answer 2

These accessory proteins can line the pore of the α subunit intertwine with transmembrane domain of the α subunit interact with the outer surface of the transmembrane domain or alternatively they can be intracellular proteins that bind to the α subunit when needed

Question 3

Which members of the KCNE family are found in epithelia

Answer 3

KCNE1-E3

Question 4

KCNE family proteins are all low molecular weight small proteins. What sort of sizes are they

Answer 4

Between 103-177 amino acids

Question 5

How many transmembrane domains do K+ channel β subunits have

Answer 5

1 transmembrane domain

Question 6

Outline how different members of the KCNE family can have different effects on the channel which they regulate

Answer 6

KCNE1 shifts the time course of activation of the channel by changing the gating properties of the channel as well as conferring its cAMP-dependence. Meanwhile KCNE2 actually converts the voltage-gated channel to a non-voltage-gated channel

Question 7

How do the currents generated by KCNQ1 change when co-expressed alongside KCNE1

Answer 7

Co-expression of KCNE1 with KCNQ1 results in much larger current generation. This occurs alongside slowing of the time course of activation so that maximum currents are reached at a longer time point

Question 8

How is a negative membrane potential and a driving force for Na+ influx set up in the PCT cells

Answer 8

The Na+/K+ATPase pumps 3Na+ out and 2K+into the cell thus creating a driving force for Na+ entry. Then the open basolateral K+ channels allow K+ ions to move back into the cell shifting the membrane potential negative towards EK

Question 9

How is the Na+ gradient in the PCT cells utilised to transport other species

Answer 9

The Na+/Glucose cotransporter brings Na+ and glucose into the cell at the apical membrane. It utilises the Na+ gradient set up by the Na+/K+ATPase to bring glucose in. Na+ brought in by the Na+/Glucose cotransporter is the pumped out basolaterally by the Na+/K+ATPase. Meanwhile glucose diffuses out at the basolateral membrane by glucose facilitated transport protein down its concentration gradient

Question 10

What species are absorbed by the PCT cells

Answer 10

There is a net absorption of Na+ and glucose. This also acts to drive a paracellular Cl- reabsorption which alongside the other solutes contributes to water reabsorption

Question 11

Below are two pieces of data looking at the expression of KCNE1 (on the left) and KCNQ1 (on the right) in the cells of the renal cortex. Describe what these pictures show

Answer 11

The staining shows that KCNE1 and KCNQ1 are expressed in the brush border of the nephron most likely in the PCT. This is indicated by the ring like-staining that can be seen in both which is indicative of the renal brush border. This corresponds to the apical membrane of the cells that line the PCT. It can however be seen that expression of KCNE1 is not identical to KCNQ1 which suggests that there isnt a total overlap in expression of the two proteins

Question 12

What is implied by the fact that KCNE1 and KCNQ1 expression don’t entirely overlap

Answer 12

There are parts of the proximal tubule where KCNE1 is regulating a different channel (not KCNQ1)

Question 13

Outline the clearance study experimental setup

Answer 13

Anaesthetise the mouse and place it on a heated pad that is controlled by a rectal thermometer that measures the mouse’s core body temperature. The carotid arteries of the mouse are then cannulated to allow the measurement of blood pressure which acts as an indicator of the depth of anaesthesia. This cannulation also allows you to take a blood sample at the end of the experiment. In addition the jugular vein is also cannulated to allow for fluid replacement as well as any infusion of drugs. Finally the bladder is also cannulated to collect the urine for analysis looking at kidney function

Question 14

What is the impact of KCEN1 knockout on Na+ and Cl- concentrations in the blood plasma

Answer 14

Knockout of KCNE1 doesn’t impact the ability of the animal to maintain Na+ and Cl-

Question 15

GFR rate is massively decrease in mice harbouring a KCNE1 knockout T or F

Answer 15

F – GFR rates are unchanged

Question 16

What interesting phenomena was observed in glucose handling of mice with the KCNE1 gene knocked out

Answer 16

Plasma concentration of glucose were significantly lower in KCNE1 knockout than wild type. This implies that there were problems in glucose handling at the level of the kidneys. However the extremely high blood glucose levels make this result questionable

Question 17

How is the fractional excretion calculated

Answer 17

The rate of excretion of a solute divided by the rate of filtration of that compound

Question 18

What does the data below show about the effects of KCNE1 knockout on the fractional excretion of solutes

Answer 18

The data shows that KCNE1 knockout mice excrete more Na+. Compared to wild type where 0.5% of Na+ being filtered was being excreted 1.5% of Na+ being filtered in the KCNE1 knockout was being excreted. This implies that the knockout mice have a problem in reabsorbing Na+ somewhere in the nephron. In addition there is the same problem with Cl- excretion with KCNE1 knockout mice excreting more Cl-. 1.25% of filtered Cl- was excreted in the wild type animals however 2.5% of Cl- being filtered in the KCNE1 knockouts were being excreted. This implies that the knockout mice also have a problem in reabsorbing Cl- somewhere in the nephron. The same effects were seen in glucose and water excretion with the KCNE1 knockouts having increased fractional excretion of glucose and fluid compared to wild type

Question 19

Subsequent data carried out on KCNE1 knockout mice found that the mice had an increased fractional excretion of Na+ Cl- and H2O. What was found in regards to glucose handling

Answer 19

This study actually found no increase in glucose excretion in the KCNE1 knockouts compared to wild type. In addition glucose levels were recorded at much more physiological values within the expected range

Question 20

What was the conclusion of the results the KCNE1 knockout mice had increased fraction excretion of Na+ Cl- glucose and H2O

Answer 20

That KCNE1 is playing an important role in the kidney regulating a K+ channel that is driving Na+ Cl- and water excretion

Question 21

In order to determine which channel KCNE1 was regulating in the PCT the KCNQ1 blocker chromanol 293B was added. What were the effects of this on Na+ Cl- and H2O excretion in wild type cells

Answer 21

Infusion of chromanol decreased Na+ reabsorption in the wild type cells. This implies that there was an increase in Na+ excretion. In addition the same effects were seen on Cl- and H2O handling both seeing increased fractional excretion implying that reabsorption was being compromised by KCNQ1 blockade

Question 22

What was seen in terms of Na+ Cl- and H2O handling in response to chromanol in the KCNE1 knockout cells

Answer 22

Infusion of chromanol into KCNE1 knockout mice had no effect on Na+ Cl- or H2O handling all of which had an unchanged fractional excretion.

Question 23

Treating wild type animals with chromanol 293B mimicked what is seen in the KCNE1 knockout mice; increased Na+ Cl- and H2O excretion. What does this tell us about the role of KCNE1

Answer 23

This indicates that KCNE1 is regulating a chromanol 293B-sensitive K+ channel that is important in driving Na+ Cl- HCO3- and therefore water handling in the PCT

Question 24

If KCNE1 in the PCT was involved in regulating KCNQ1 what would be seen in the knockout mice for both genes

Answer 24

If KCNE1 regulates KCNQ1 then knockout of KCNE1 and KCNQ1 should results in the same/similar phenotypes (increased fractional excretion of Na+ Cl- and H2O compared to wild type)

Question 25

What was the effect of KCNQ1 knockout with regard to Na+ and H2O handling in the PCT. What does this suggest about the role of KCNE1

Answer 25

The fractional excretion of Na+ and H2O was no different in the KCNQ1 knockout compared to wild type. Hence under normal conditions the KCNQ1 knockout was very different from the KCNE1 knockout. This suggests that KCNE1 isn’t regulating KCNQ1

Question 26

Below is some patch-clamp data looking at the chromanol-sensitive currents generated by wild type PCT cells and those with KCNE1 knocked out compared to experimental currents recorded in expression systems expressing KCNE1 and KCNQ1. This was used to determine if KCNQ1 is indeed the channel regulated by KCNE1. Describe what this data shows

Answer 26

The graphs shows that there are no chromanol-sensitive currents generated in the KCNE1 knockout mice implying that when KCNE1 is missing the channel can’t function. However the chromanol sensitive currents generated by wild type PCT cells don’t look like KCNQ1 + KCNE1 co-expression currents. This implies that the channel being regulated by KCNE1 in the PCT is unlikely to be KCNQ1. Due to the fact that chromanol can inhibit other K+ channels it may be possible that in the proximal tubule there is a chromanol sensitive K+ channel that is regulated by KCNE1 is not KCNQ1

Question 27

Below is a model of the channels involved in the secretion of acid by the parietal cells in the stomach. Describe the region of the model depicted in the red box

Answer 27

CO2 and H2O move into the cell at the basolateral membrane where once inside the cell they combine to form carbonic acid. Carbonic acid then dissociates into HCO3- and H+. HCO3- then recycles over basolateral membrane in exchange for Cl- which in turn is secreted at the apical membrane. In addition there is an apical secretion of K+ through K+ channels. This K+ recycles through the K+/H+ATPase in exchange for H+ secretion. This is how the parietal cells secrete acid and lead to acidification of the stomach contents

Question 28

What mechanisms are there for stimulating acid secretion by the parietal cells

Answer 28

Parietal cells are stimulated to secrete HCl by basolateral receptors. This includes acetylcholine acting on M3 receptors histamine acting on H2 receptors and gastrin acting on CCKB receptors

Question 29

Without the apical K+ channel in the parietal cells there is insufficient apical K+ to drive H+ secretion T or F

Answer 29

T

Question 30

The activity of which transport protein can be determined from the ammonium pulse experiments

Answer 30

The H+/K+ATPase which is driven by apical K+ secretion

Question 31

Outline the ammonium pulse technique

Answer 31

Ammonium is added to the parietal cells which then dissociates in the extracellular fluid into H+ and NH3. NH3 can then diffuse in to the cells where once inside it binds to H+ and shifting the pH more alkaline. Then removal of the extracellular solution reverses the driving force as NH4 is now removed from extracellular solution. This causes NH4 inside the cells to dissociate back into H+ and NH3 before the NH3 then diffuses back out of the cells. The H+ ions left behind inside the cells causes an acidification which in turn leads to the upregulation of mechanisms to secrete acid

Question 32

Which transport protein serves to mediate the recovery from acidification that occurs after NH3 diffuses out of the cell

Answer 32

The H+/K+ATPase

Question 33

What is seen in the ammonium chase experiments carried out in parietal cells with the KCNQ1 gene knocked out

Answer 33

There is no recovery from acidification that is the result of no functional KCNQ1 protein. This means that it is likely that this is the apical K+ channel responsible for driving K+ secretion which in turn drives H+ secretion

Question 34

Hence which channel in the apical membrane of parietal cells in the gut is responsible for setting up the driving force that leads to H+ secretion and acidification of the lumen

Answer 34

KCNQ1

Question 35

Below is some data investigating the effects of KCNE2 knockout on stomach pH with and without the presence of histamine compared to wild type. Describe what this data shows

Answer 35

This data shows that wild type stomach pH is around pH 4 but that the addition of histamine causes an acidification of stomach pH shifting it to around pH 2.8. In contrast the KCNE2 knockout mice have a more alkaline stomach with a pH of around 6. Interestingly the addition of histamine to KCNE2 knockouts parietal cells results in no shift in stomach pH and no acidification. This implies that they are struggling to secrete acid in response to histamine which in turn suggests that regulation by KCNE2 is required for this.

Question 36

What phenomena is seen in KCNE2 mice regarding the levels of particular circulating gut hormones

Answer 36

KCNE2 knockout mice are achlorhydric as they are struggling to secrete acid. However circulating gastrin levels are higher this is an attempt to compensate for decreased acid secretion through the apical K+ channel

Question 37

What does this ammonium chase data show about the effects of KCNE2 knockout on acid secretion

Answer 37

This data shows that KCNE2 knockout mice are unable to recover from acidification of the cells by stimulating acid secretion. This data looks very similar to the data for the KCNQ1 channel indicating the KCNE2 may be involved in regulating KCNQ1. The lack of ability to recover from acidification in KCNE2 knockouts suggests there is insufficient K+ secreted into the apical solution by KCNQ1 to drive H+ secretion. This in turn implies that KCNQ1 and KCNE1 seemingly work together to mediate the K+ secretion required for acid secretion

Question 38

What feature of the knockouts of KCNE2 and KCNQ1 suggest they act together to secrete acid in the parietal cells

Answer 38

The phenotypes of a KCNQ1 and KCNE2 knockouts in the parietal cells is seemingly the same

Question 39

Which β subunit regulates KCNQ1 in the parietal cells

Answer 39

KCNE2

Question 40

Which β subunit regulated KCNQ1 in the PCT

Answer 40

It is still unknown