Lecture 9

Question 1

what are the features of beta subunits

Answer 1

• beta Subunits Cytoplasmic/ Integral membrane proteins
o Can be clustered up with the helices of the alpha subunit
o Trafficking, regulation and open probability
o Normal function of  subunits/ ion channel – only seen If you have  subunits

Question 2

what are the different members of the KCNE family?

Answer 2

• KCNE1 – 103-177 amino acids (prev mink)
o Regulates Q1 K channel - KCNQ1 (Long QT syndrome)
• KCNE2 – 1 TM domain
• KCNE3 – Excitable – long QT syndrome
• KCNE4
• KCNE5

Question 3

what are the effects of KCNE1 on KCNQ1?

Answer 3

oQ1 channels larger
oE1 enhances currents/function of Q1
oDouble in current magnitude
oShift in time dependence - without E1 Q1 reach steady state faster

Question 4

where is KCNE1 found?

Answer 4

found in apical membrane of proximal tubule of the mouse kidney

Question 5

how are in vivo clearance studies undertaken?

Answer 5

• Aneasthetise mice and place on heat pad – allows mouse to maintain normal body temp
• Thermometer in rectum – if core body temp drops a little sends signal to heat pad up
• Cannulate carotid artery - Measure blood pressure
o need good bp for you to be able to use them
o also determines depth of anaesthesia
o pinch test – reflex response = need more anaesthetic
• Cannulate jugular vein – fluid replacement – saline
• Cannulate bladder –
o collect urine – volume per unit time - analyse composition
• composition of blood and urine allow us to look at renal function

Question 6

what occurs in KO KCNE1 studies?

Answer 6

Same plasma conc of Na and Cl, glucose is slightly higher.
No change in glomerular filtration rate.
Low FE - meaning lots of reabsorption o Glucose - higher in KO - not as big of a difference
o Cant reabsorb as much water – also higher in KO = lose more fluid – increased urine flow rate
o Mainly losing Na, Cl and water in urine
o Increased urine flow rate
o Driving force for Na uptake reduced – in urine

Question 7

what is the effect of chromanol 293B?

Answer 7

inhibits KCNQ1 – mimic losing E1
Turned WT into KO mimic
No effect to KO - Lack of E1 – means Q1 not working anyway
Increase FE in Na, Cl water in the WT

Question 8

what did Q1 KO clearance studies show?

Answer 8

FE – exactly the same in WT and KO

E1 in proximal tubule unlikely to regulating Q1 K channel

Question 9

when looking at chromanol sensitive studies, what does E1 KO show?

Answer 9

o No function of K channel – no chromanol sensitive currents
o Losing E1 stops channels from functioning, leading to the symptoms we see in struggling to set the membrane potential and struggling to reabsorb Na, Cl and water

Question 10

what is the gastric function of KCNE2 & KCNQ1?

Answer 10

• Acid secretion in parietal cells:
o CO2 and water move into parietal cells and under the influence of carbonic anhydrise -> production of bicarbonate and H+
o Bicarbonate leaves across basolateral membrane in exchange for chloride
o Increased Cl conc in parietal cells – so if you open up channel you get secretion of Cl – as HCl
o K/H ATPase
• Uses ATP to exchange K coming into the cell for H+ leaving
• Loss of H -> Net secretion of HCl
• Movement of k out of cell drives H secretion
• If no K coming out of the cell – there is insufficient K outside to support ATPase
• Important to maintain acid secretion

Question 11

what receptors are important for acid secretion?

Answer 11

o Ach – M3 muscarinic receptors
o Histamine -activates H2 receptors
o Gastrin – CCKB receptors

Question 12

how is gastric function tested?

Answer 12

Ammonium pulse technique – NH4+

Question 13

what are the steps involved in the ammonium pulse technique – NH4+?

Answer 13

o Ammonium -> ammonia -> H+
o Ammonia goes into cell and mops up H+ inside cell
o pH goes up – alkaline
o take away ammonium from outside cell
o ammonia dumps hydrogen ions and goes down conc gradient and leaves the cell
o acidification
o look at rate of recovery of pH

Question 14

what did KCNQ1 KO in gastric function studies show?

Answer 14

Q1 KO – no recovery of pH – until Na added -not able to secrete K
Parietal cells KO secrete less H+
Due to problem apical K+ secretion

Question 15

what was the effect of KCNE2 KO on gastric function?

Answer 15

oHeterozygous -> have 50% normal E2 – so response fairly normal
oHomozygous -> complete E2 KO – stomach v acidic before histamine, addition of histamine doesn’t have an effect – cant secrete any acid
o lack of function of K/H ATPase, Parietal cells KO secrete less H+, Due to lack of function of K channels as E2 has been knocked out

Question 16

what is the effect of KO on Q1/E1 on the apical membrane on acid secretion?

Answer 16

oKO of either – K secretion doesn’t occur
oInhibits H secretion
oInhibit secretion of HCL by parietal cells in stomach

Question 17

A patient has a mutation in KCNE3. What is the impact of this mutation in terms of upper airway cells and what symptoms might be observed?

Answer 17

• NKCC1 – TRANSPORT PROTEIN – not ion channel!
• E3 is on basolateral membrane of upper airway
• Regulates Q1 K channel – set negative membrane potential
• Transport Na, K, Cl into the cell
• IC Cl not as
• CF similar symptoms
• Lof mutation –
o Q1 doesn’t function normally
o Depolarisation of basolateral membrane potential
o Reduced driving force for NKCC1
o Less IC Cl-
o Reduced driving force for Cl secretion
o Less Cl secretion
o Mimics CF – So increased risk of infection, lung damage and