L9-B subunits Flashcards
what is an alpha subunit?
the subunit/protein which makes up the ion channe
what is a beta subunit?
subunit/integral membrane proteins with a small MW which interact with and regulate the activity of alpha subunit.
- the modify the properties of alpha subunits
Which members of the KCNE family are expressed in epithlial cells?
- KCNE1-3
What effect does KCNE1 have on KCNQ1 and how was this measured?
KCNE1 causes larger currents and enhances Q1 function
- there is changes in the gating and reaching a steady state is slower/delayed
- this was determined by 2 electrode voltage clamp techniques in xenopus oocytes expressing cRNA encoding fo Q1, or Q1 and E1 (overexpression)
what role does KCNE1 have in renal funtion?
- main role is in proximal tubule
- sets negative resting potential which creates driving force for Na and glucose to enter the cell via apical membrane
- net absorption of Na+ , Cl- and water follows
- E1 expressed in apical membrane- is there also a K+ channel on apical membrane?
What is relationship between location of E1 and Q1 on the apical membrane of the proximal tubule?
There are some places where epxression overlaps but is no direct overlap
- is there another K+ channel that E1 is regulating?
give the channels on a simple cell membrane of a proximal tubule renal cell
-apical - Na+ and Glucose pump - into cell
basolateral- Na/K+ ATPase
basolateral - K+ channel - out of cell
basolateral - glucose out of the cell
how were the mice treated in the Q1/E1 KO studies looking at kideny function?
- mice were anaesthetised and put on a heat pad
- jugular vein was cannulated - fluid replacement
- carotid artery cannulated - BPmeasurments and blood smaple - indicate depth of anaesthia and BP has effect on kidney function
- cannulate bladder - collect urine for analysis
what effects did KO E1 have on blood levels in the first exp?
KO E1
- no change in plasma [Na+] or [Cl-] (E1 doesn’t impact on animals ability to regulate Na, Cl in plasma)
- [glucose] in KOE1 animal is lower than in WT animal (problem in glucose handling at the kidney level) - but glucose levels were abnormally high (125mM, should be between 5-10mM) - confidence in paper is limited
- no significant difference in glomerular filtration rate
what is the equation for fractional excretion (FE)?
- Rate excretion/ Rate filtration
- if 100%, everything being filtrated is being excreted
what did another exp show which looked at the effects of E1 KO on the kidney?
- KOE1 - animals lose sodium - reduction in sodium reabsorption (higher FE)
- KOE1 - animals lose chloride - less CL- reabs (higher FE)
- KOE1 - animals lose glucose - less glucose reabs (higher FE)
- loss of water - higher urine flow rate - less water reabs as it follows Na and Cl (higher fluid FE)
- in this exp the plasma glucose was around 10mM which is within the normal/epxected range
- shows E1 is a subunit regulating a channel somewhere in the kidney which plays an important role helping the kidney drive NACL and glucose reabsorption
what does newest data looking at E1 KO i the kidney show?
- E1 KO there is reduced Na+, Cl- and water reabs
- BUT no difference in glucose handling/reabs like other exp showed
- different backgrounds?
- still OK comparison
what does chromanol 293B inhibit?
Q1
What happens when chromanol 293B is added to WT and E1KO cells?
- infusion of chromanol 293B into the jugular vein
- 293B turns WT values into E1 KO values (reduced Cl, Na and water reabs)
- 293B has no effect on E1 KO
- shows E1 is regulating a 293B sensitive K+ channel (not definately Q1)
if there is a lack of effect that E1 has on glucose handling what does this suggest its location is?
LATE proximal tubule
What happens to kidney function when Q1 was knocked out of mice, and what does this show us?
- When Q1 was knocked out of mice this data looked different to when E1 was knocked out
- in Q1 KO is a decrease/no difference in Na+ or H2O excretion)
- this suggests that E1 is not regulating Q1, but is regulating another chromanol sensitive K+ channel
What was shown by patchclamp studies measuring chromanol sensitive currents in E1KO and WT animals?
- in E1KO mice, the chromanol senstive current in 0
- In WT mice the chromanol sensitive current is higher than 0
- in E1/Q1 mice the chromanol sensitive current is the highest
- this shows that the K+ channel regulated by E1 in the proximal tubule is not Q1 as has different 293B sens currents to Q1/E1
- E1 is regulated a different K+ channel which is 293B sensitive
what can stimulate acid secretion?
histamine
Ach M3
Gastrin
give channels on the cell model of a parietal cell?
apical- Cl- secreting channel
apical- water moves out
apical- K+ channel moves out of cell
apical - Potassium proton ATPase - K+ into cell, H+ out of cell
Basolateral - Na/K+ ATPase
basolateral - K+ channel out of cell
basolateral - Hco3- out of cell, Cl- into cell
basolateral - CO2 moves through membrane into cell - combines with waters OH- to form bicarbonate
basolateral - water moves into cell dissociates, and H+ moves through K+/H+ exchanger
basolateral - H+/Na+ exchanger, Na+ into the cell , H+ out of the cell
ACID SECRETION INTO LUMEN OF STOMACH
what technique was used to examine Q1s role in gastric function?
ammonium pulse chase technique
- expose cells to ammonium
- dissociates into Nh3 and H+and NH3 moves into cell - combines with H+ - more alkali
- the NH4+ already in cell - separated into NH3 and H+
- when ammonia moves out of the cell - leaves H+ ions- acidification
What happens to gastric function when Q1 was KO?
when Q1 KO there is no acid secretion in parietal cells
- there is no H+ secretion via the K+/H+ ATPase
- This is because there is less K+ moving out of the cell into the lumen via Q1 - so there is less K+ to move back in and drive H+ secretion
- cells cannot recover from acidic state when ammonia removed - as cannot secrete H+ ions
- exp conducted in absence of Na/H+ as this would effect results
why were exp examining KOQ1 on gastric function performed i the absence of Na/H+ exchanger
this would interfere with results as at acidic PHs H+ could diffuse out of the cell via NA/H+ on basolateral membrane
- would effect PH and would not representable of acid secreiton into the lumen
what is achlorhydra?
Where HCl secretion is absent or low
What happens to gastric function in a E2 KO mouse and how was this shown?
- in E2 KO mouse stomach PH is higher even with histamine stimulation (no response)- shows cells are struggling to secrete H+
- In Wt upon histamine stimulation, H+ secretion - stomach PH falls
- using ammonium pulse technique, E2 KO shows no recovery of acidic conditions
- heterozygous shows slow, but some recovery
- shows parietal cells of E2KO secrete less H+
- also is high circulating levels of gastrin in E2 mice as are trying to stimulate acid secretion
