B Sub unit regulation

Question 1

What are B sub units with regards to K+ channels?

Answer 1

Proteins that modify the properties of a sub units
Can be located at the membrane or the cytoplasm
Interconnected with helices at the alpha sub unit of channels

Question 2

KCNE FAMILY - what do the relevant sub units regulate?

Answer 2

KCNE1, 2 and 3 regulate the K+ channels found in the epithelia and regulate KCNQ1 – they are excitable, consist of one TMD

E1 mutated in Long Q-T syndrome!!

Sub units have different effects on the channel (in terms of voltage dependence etc) - determines how channels function under normal conditions!!!

Question 3

KCNQ1 and its relationship with E1?

Answer 3

KCNQ1 is the ion channel and E1 is the sub unit that regulates it

Question 4

Results of voltage dependant current when xenopus oocyte expressing cRNA encoding KCNQ1 only AND both KCNQ1 and E1

Answer 4

With overexpression of E1, the WT currents produced were larger, had slower activation and a shift in voltage dependence was seen

Question 5

Where was the majority of E1 localised to (area of nephron and membrane?)

Answer 5

Majority seen in the proximal tubule and on the apical membrane. Some Q1 seen in the distal nephron and E1 / Q1 expression not the exact same

Question 6

In clearance studies of KO Q1/E1 mice, what is cannulated and for what reason?

Answer 6

• Jugular vein - measure blood ion concentrations
• Bladder - measure urine ion concentrations
• carotid artery - measure blood pressure and blood ion concs

Question 7

In clearance studies of KO E1 mice, what are the impacts on plasma K+/CL-, GFR and plasma glucose conc?

Answer 7

• No difference to plasma K+/Cl-, v similar GFR to wild type and a (unusual) higher plasma glucose concentration

Question 8

In clearance studies of KO E1 mice, what are the impacts on FE? (robson paper)

Answer 8

Increased fractional excretion of Na+, Cl- and H20 (there is a struggle in the re-absoprtion of these ions in the absence of E1), increased urine flow rate –» this is a good indicator that there has been change in the tubular function

Question 9

What do the results of the clearance studies from the KO E1 mouse suggest about the loss of E1?

Answer 9

Loss of E1 = loss of functioning of KCNQ1 channel –> loss of channel contribution to the membrane potential = decrease in the driving force for absorption of Na+/ Cl-/ H20

Question 10

KO E1 mice with and without c329B (Q1 channel inhibitor) – what were the impacts seen?

What does this show?

Answer 10

The increase in fractional excretion seen upon addition of the KCNQ1 channel blocker in the WT and KCNE1 knockout mice was around the same –> inhibiting the channel and knocking out E1 was having the same impact!!

E1 is regulating a chromanol-sensitive K+ channel

Question 11

KCNQ1 mouse knockouts - what was the impact on fractional excretion?

Answer 11

There are some differences in glucose handling but under normal conditions there is no impact in the fractional excretion of ions – doesn’t mimic E1 knockout – this shows that E1 is not regulating the Q1 channel in the kidney!!

Question 12

Patch studies of total whole cell currents in the proximal tubule with Chromanol added –> the decrease in current showed properties of channels / channel profiles!

How is this consolidated by KO mouse data?

Answer 12

Voltage dependant K+ channel chromanol sensitive current profile very different to what is expected from proximal tubule – E1 is regulating a different K+ channel!

Consolidated by same experiement in KO Q1/E1 animals - no currents at all –» no chrom-sensitive K+ currents in the proximal tubule that don’t have E1!!!!!

Question 13

Main functions of E1 in the proximal tubule?

Answer 13

• KCNE1 needed for Na+/Cl-/HC03- handling
• Probably in proximal tubule but late (little impact of KO on glucose handling suggests that it impacts late PT as there is little glucose handling here)
• Role in maintenance of the membrane potential – transport function!!!

Question 14

KCNQ1/E2 and its link to gastric function – how is acid secreted and how is this gradient maintained?

Answer 14

H+ secreted into the stomach in exchange for an K+ back in by H+/K+ ATPase pump on the apical side of the epithelial cells.
The K+ is recycled back out across the apical membrane via a K+ channel as there is not enough in the stomach to support the action of the H+ pump

Question 15

KCNQ1/E2 and its link to gastric function – how is Cl- secreted and how is this gradient maintained?

Answer 15

Cl- is secreted at the apical membrane via a Cl- channel - this gradient is generated by the HCO3-/Cl- exchanger on the basolateral membrane.
This means that Cl- is above its electrochemical gradient and is secreted via a channel

Question 16

KCNQ1 K.O – ammonium pulse technique - what does this technique measure in cells?

Answer 16

Measures H+ secretion in cells and therefore is used as a measure of PH recovery under shock changes to PH in cells / cells ability to recover from changes in PH

Question 17

What was shown in KCNE2 KO cells ??

Answer 17

No PH recovery seen in E2 KO cells –> lack of function of this apical K+ channel meant that the apical H+/K+ ATPase could not work as there was no sufficent supply of K+ in the stomach outside of the cell (which is mediated by the action of this K+ channel) –> gastric endothelium couldn’t secrete H+ therefore and no PH recovery seen.

Question 18

What ligands can stimulate H+ secretion at the level of the stomach and what receptors do they bind to?

Answer 18

• Ach - stimulates secretion via muscarinic receptors
• 5HT - via H2 receptors
• Gastrin - via CCUB receptors