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1
Q

Experiment to show that Enac has a function in controlling height of ASL

A
  • Add excess fluid
  • Mimic in vivo conditions of breathing- moving back and forwards
  • look at rate of decrease of height due to change in trans epithelial potential due to changes in Cl secretion and Na reabsorption
  • Butamide and Amiloride inhibition over time
  • 0 hr lots of Amiloride inhibition but decreases to 30% after 48hours
  • Ohr less butamide is 55% inhibition at 48hours
    shows more Cl secretion needed to maintain ASL than Na reabsorption but both help maintain the layer
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2
Q

RSV experiments

A
  1. Look at the effect of RSV on amiloride sensitive ssc
    - Add amiloride
    - then add either control or RSV for an hour
    - then re add amiloride and see if there is a change in deflection
    RSV= decrease deflection of amiloride as less enac is functioning
  2. Look at which pathways of ENAC RSV interacts with
    - PKC (BIM inhibitor), Glycoprotein (NA inhibitor), Glycolipid (PMCD)
    - If you add inhibitor then there will be no effect of amiloride as the enac is already blocked- PKC AND GLYCOLIPID
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3
Q

Experiments for Influenza

A
  1. prove that M2 causes long term influenza
    - Overexpressed Enac and it inhibited Enac
    - decrease open probability of Enac- less deflections when M2 is overexpressed
    - caused endocytosis- western blott showed less alpha subunit and Beta subunit present when M2 overexpressed
    - showed there was less enac due to increased endocytosis- used liddles where endocytosis cant occur and showed that M2 couldn’t endocytose when liddles occurs
  2. look at mechanism of how M2 acts on enac
    - immunostaining- RFP-M2 Or RFP alone
    - ROS= green
    Results- in areas overexpressed with M2 (red) there is also green staining so ROS has accumulated
    Part 2- prove ros is involved
    - inhibited ROS with GSK- antioxidant that scavenges ros
    - also inhibited PKC by Inhib and GO6967 which blocks
    - Inhibiting PKC or M2 expressed more Enac
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4
Q

How is Cl secreted?

A
  1. Experiment to show DF created- look at rectal shark model
    - oubain- Na/K ATPase inhibitor- reduces NKCC and therefore Cl secretion as cant set up the df. Maintains low IC Na
    - Barium- blocks K- hyperpolarises the cell, Vte -1 to 0- DF for Cl secretion
    - Furoesmide- NKCC inhibitor- stops Cl influx into cell for CFTR to secrete
  2. Above electrochemical gradient
    - no active process then ic would be the same as EC
    - we got 17mM but actually 70mM as active process causing accumulation of cl- this is Na/K ATPase
    - above electrochemical gradient means Cl channels just have open for CL secretion
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5
Q

Experiment to look at mechanism of CFTR

A

Isolated rat CFTR
looked at the water content- which is dependent on Cl secretion by CF- Secrete Cl and water follow
PGE2- Increases cAMP- increases CFTR- increases Cl secretion
Ach- increases IC Ca- activates apical and basolateral Cl

  • Open CFTR= Cl secretion
  • Open K channel= hyperpolarises
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6
Q

Experiment to look at the activation of CF and Non CF

A

Ussing chamber experiment
Look at effect of cAMP secretion
- indomethacin- inhibits PG- decrease cAMP
- Imbx- inhibits phosphodiesterase- increase cAMP
- CCB- increase Ach- increase cAMP
- forskin- increase AC- increase CAMP

block amiloride to stop contamination

  • stimulate with ccb- increase Cl secretion- increase neg shift Vte
  • CCB +indomethacin- block cl secretion- DF there but channel is closed
  • CCB+ indomethacin+ imbx- bypass and increase Cl secretion

*CF patient no effect= CFTR channel is not functional

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7
Q

What experiment would you include in VX770 and G551D

A
  1. Drug screening
  2. Rat thyroid- add VX770 +FSK +G551D
  3. Expression system- Vx770 + PKA and ATP
  4. Look at Vx770 in native cell- Human bronchial cell- amiloride sensitive ssc in double mutant when adding 770 and FSK
  5. Effect of 770 on ASL layer- add fluid and VIP (increase CAMP) and see how 770 increases ASL
  6. Look at how 770 effects CBF
  7. clinical trial in humans- G551D and 770- FEV1%- improvement, pulmonary exacerbation and sweat cl
  8. G551D and Vx770 in Human nasal cells, quirt in Cl free solution and isotopreneral to secrete B agonists and increase CAMP- look at Cl secretion
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8
Q

Experiment to look at F508

A
  1. drug screening- compounds that drive immature to mature
  2. F508 and VX809 ratio of mature to immature
  3. pulse chase experiment HEK and CFTR - amount of mature and immature depends on the length of time cells are exposed to vx809
    4.
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9
Q

How do you screen for G551D and F508

A

G551D- Increase in fluorescence vm assay when increase membrane potential- which means gating worked

  • tested 228,000 chemicals
  • look at nerst= Cl
  • found VX770

f805- immunoblot assay to see which chemical drive CFTR glycosylation and become mature

  • see 2 bands
  • glycosylated is higher MW band
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10
Q

Look at the effect of VX770

A

Rat thyroid cells
stimulated G551D and control with FSK (increase cAMP)
G551D + FSK- small increase in isc
GSS1D+ FSK + 770- increases currents of cl secretion- 12-15% of wt- only needs to be that to get normal function
Prove it was CFTR that increase Cl secretion by inhibiting

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11
Q

Expression system 770

A

In vivo
- look at the effect of PKA and ATP looked at deflections

G551D- few deflections even in presence of PKA and ATP
G551D + 770- channels open, potentiator- increase Cl secretion

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12
Q

Effect of 770 on human cells

A 
Human bronchiole epithelial cells 
Block amiloride= contamination 
- severe CF- contain 2 mutations in G551D/F508 
- add FSK to stimulate 
WT- 56uA FSK induced ssc 
G551D/f508 +fsk= 2.9uA
mutant + FSK + 770= 27uA= 48% of wt 
enough to alleviate the symptoms of CF
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13
Q

Looking at the effect of 770 on ASL

A

Amiloride to block enac
added excess fluid to the layer
Used VIP- increase cAMP

WT- add excess, stimulate with VIP and the layer decreases then plateaus
G551D +VIP- massive decrease in liquid layer
+ 770- increase ASL- in-between wt and mutant

Looked at the effect on CBF
decrease ASL= decrease CBF
- G551D + VIP- decreased CBF- struggle to beat- restored when liquid is added on top
+779- restores normal layer

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14
Q

Clinical trial for 770

A 
83 patients with CF- mutations in atleast 1 CFTR 
random double blind and placebo 
- Looked at FEV1% 
- Pos= improvement 
neg= worsening

Results
- add VX770 to G551D
FEV1% Improves drastically over 2 weeks and whole time whereas placebo worsens
- Pulmmonary excabertion- Ivacaftor- 67% haven’t, placebo- 40% haven’t
- Sweat cl- drops below threshold

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15
Q

Part 2 of Trial

A

Inserted Cl free solution containing Isoprenterol (increase cAMP) into nasal passage- initiates Cl secretion
CFTR working then there is a negative shift in potential as more Cl secretion occurring
VX770- 75 and 150mg- increased Cl secretion
direct experiment

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16
Q

Looking at the function of F508 and VX809

A

Looked at ratio of mature to immature
no 809- 0.5%
increased by 30% when VX809 was added

Works by inserting in protein of CFTR and correcting folding so it can be transported to the membrane rather than for degradation

17
Q

Pulse chase experiment Hek and CFTR

A

35s methionine and 35s cysteine
mature cftr
35s removed and compounds left for a duration of time
- formed ratio of Mature Glycosylated 35CFTR and immature 35CFTR
- amount of time left determines ratio

Look at western blot
- cells left for 180 min, radiolabelled immature CFTR
wt= mature shifted, decrease immature
F508- no immature and no mature- degraded
F508 and vx809- slightly more mature but not as much as wt

18
Q

Clincal trial f508

A 
19-19 CF patients 
28 days 
different amounts of F508 homologous 
Looked at FEV1%- NO PATTERN 
Highest sweat cl decrease was still above the cf threshold
19
Q

Combination therapy

A

Combining VX770 and 809
809- not sufficient to treat CF alone
1. change in sweat with 250mg there was a large sweat decrease but none below threshold
2. FEV1% increased by 3%- placebo slowly worsened
3. clinical trial
- 1108 patients, lumacaftor and ivacaftor
placebo, 6000mg + 250mg, 400mg +250mg
- Fev1% improved in first 2 weeks then steadied out, big range of results
- Cough or infection catching- decreased from 40% placebo to 20% ivacaftor
- hospitalisation- decreased
- forrest plot- FEV1%- above black line- better, below- worse, neither

20
Q

Gene therapy

A

Thought they would have done this in 1990s but still hasn’t been done due to the renewal of cells
- Example asthma- gene therapy works until cells are replaced and the new ones wont contain the new
PGM169
- FEV1% Dramatically improved
FVC- lung function improved

Doesn’t improve or worsen- stabilises- if get someone with high lung function it could work well

21
Q

Experiments for A typical CF

A
  1. Show GOF epithelial Na channel- look at mutated ENAC and add amiloride and show similar to Mild CFTR
  2. Prove aW34R was a GOF mutation- bigger function of Enac when was out amiloride
  3. See if aw349r mutation has negative feedback- looked at slightly higher amiloride
  4. Look at the effect of cleaving channels on GOF enac
  5. Self inhibition of Na due to increase Na currents- decreases channel Po
  6. MSET technique to measure Po rather than Patch clamp
  7. Effect of BV348M mutation in Atypical enac
  8. Mouse overexpression B subunit effect on survival
22
Q

Validating ENAC GOF

A
  1. looked at CF, Atypical CF and Normal vte
    - add amiloride- See function of Enac
    - atypical carrier 025mv to -15mv
  2. prove aW345r is GOF
    increase amiloride sensitive currents- increase enac function
    - bigger deflection- bigger function of enac when wash out
  3. to see if aW345R is due to negative feedback
    - started with same number of channels
    - did high and low
    - in wt low- wait for a bit before enac pulled out of membrane so slightly higher amiloride,
    -high Na was the same for WT and mutant- ratio drop same, so not a problem in endocytosis
23
Q

Effect of cleaving on open probability

A

Cleave CFTR- increases Po- more enac work
chymotrypsin= cleaves proteins
WT- add chymotrypsin, increase cleaved ENAC, increase Po,
Know its Enac cleaved as when you add amiloride this decreases
Mutant- response to chymotrypsin is lost, gating looks different but increase PO

Looked at pattern of activity- deflections
WT- add chymotrypsin- increased open probability so increased deflections
Mutant- already has lots of deflections so Po high- irrespective of chymotrypsin

24
Q

Na self inhibition

A

Self inhibition occurs- see spike in Na current as there is too much Na influx so Na channels decrease Po
this is when 100mM of Na is added
Amiloride inhibits this
Mutant- unable to self inhibit, lost even when Na is added

25
Mutation in B subunit to increase ENAC GOF
1. B subunit change in open probability - wt= increase current when take away amiloride - mutant GOF increases by 40-45% from mutation higher Po - increase Na inclux 2. SCNN1B to see effect on survival of mice Atypical CF- overexpress B subunit of ENAC, rate limiting step in Enac function Add this to channel the PCL layer decreases 1. survival curve- decrease surivial of mice overtime as cant excrete mucous 2. lung slice- plaques and plugs- trap gas in 3. When you inject the mouse with bacteria - leave 2 days wt= gone mutant- still infected as cant get rid of mucous
26
Experiments for K channels
1. Look at effect of Chromanol 293B on KCNQ1- PT-PCR 2. Ussing chamber K channel- look to see if Q1 is cAMP activated- added IBMX increased cl secretion and look if barium selective- K recycles across cell line= control amount of Cl but is is Q1 selective 3. How 293B and Ba effect CF and Non CF 4. Apical UTP on CaCC- cl secretion- Ca activated 5. Basolateral K channel secretion HSK4 using K channel blockers and UTP 6. Apical K channel- BK, inhibit using praxiline 7. Effect of Apical K channel-BK on CBF
27
Q1 channels- 293B inhibition and cAMP activated
1. Looked at fresh nasal epithelium, prove that 293B inhibits Q1 - RT-PCR - BP mw 788bp - Q1 expressed in both wt and CF- expressed in the upper respiratory tract 2. Looked at the effect of cAMP on K channel activation - Vte against time - added IMBX gradually increasing the conc- Vte became more positive as Cl secretion was occurring - block K channel= block Cl secretion 3. Is Q1 barium sensitive - inhibited ssc with 10mg of 293B- didn't go to 0 - barium takes to 0 - K recycling controls CL secretion 4. Looked at the effect of 293B in CF and Non CF - under control and IBMX effect on K and cl Non CF- when you increase cAMP in 293B - increase Cl secretion CF- even if you increase cAMP there is still no secretion as CFTR channels not functional 5. barium sensitive - non CF and CF- increase imbx- increase Ca had no effect on Ba sensitivity- cl secretion is driven by Ba sensitive K channel ontop of Q1 in CF- additional Cl channel
28
Ca effect on Apical channels
UTP increased with Increased IC CA activates Ca activated CaCC channel- initates Cl secretion from apical increase ssc as upregulated in CF patients however this is not sufficient to alleviate the CF symptoms - increase UTP- increase PGE- increase cAMP - hyperpolarising effect as it increases cAMP- increases k efflux from Q1- neg membrane potential- increase CL secretion
29
Effect of UTP on basolateral Cl channels
1. All amiloride and no cAMP - effect of 239b and clotrimazole on amiloride - UTP stimulated increasing IC cCa wt- high cl secretion 293B- no effect, cAMP low so Q1 not open clotrimazole- blocked UTP Ca activated K channel- Cl secretion decreased 2. All amiloride and cAMP - effect on CF and nonCF - wt- 293B now has an effect as channel activated through cAMP - CF- enhanced Cl secretion 293B contributes to DF in Cl from Ca activated channels
30
Apical K channel
1. inhibit paxiline- Bk inhibitor and see the effects on Cl secretion control- add atp- increase Cl secretion Paxilline- Add ATP- decrease Cl secretion 2. KD- BK inhibits Cl secretion by CaCC
31
BK on CBF
NHBE cells- lefts for 4.5 days- CBF measured- add PBL- CBF measured -CBF - T- no addition to ASL layer- paxiline reduce asl R- restored BK - T - CBF non existent as KD K= not K efflux= no Cl secretion - R- restores
32
B subunit experiments
1. Effect of overexpressing Q1 and E1- patch clamp and voltage dependence 2. KCNE1 KO mouse and renal function- kidney- Q1 thought to be apical 3. Clearance study to see the effect of KO on amount of GFR, plasma conc and glucose 4. is Q1 regulating E1?- KO Q1 293B and if E not working then when you block Q1 there should be no change but there is a shift in FE1% NA 5. Patch clamp- show its not voltage dependent
33
What does the KCN31 and KNCE2 study show
Stimulants Ach, histamine, gastrin Secretion of HCL from stomach digests food in stomach before small intestine - Apical Cl open and releases Cl bicarbonate- not enough K to support this 1. Ammonium pulse technique - Ammonia in cell, combine with H, alkylation, increase PH then take away ammonia releasing H ions and an acidification lack of recovery= KO wt- recover from PH increase exchange for H channels 2. Parietal cell KO KO= decrease h KO mice increase PH- more alkaline struggle to secrete acid and not as much in stomach
34
Ammonium pulse technique on KNCE2 KO in gastric function
WT- recover HETs- secrete some acid Hom- no acid secretion- E2 not working so cant regulate their Q1
35
Relationship between E1 and Q1
1. They looked at patch clamp technique - 2 electrode voltage clamp in xenopus ooctyes expressing cRNA encoding E1 and Q1 - thought that E1 modifys/interacts with Q1 2. Voltage dependence- looked at lines between in Q1 and Q1/E1 - saw that currents activity was greater - slower activation - still voltage dependence 3. E1 ko study in renal mouse- looked at kidney function - immunostaining showed that apical E1 and nuclei and where they thought Q1 was stained - turned out that Q1 is apical and E1 is proximal
36
Clearance studies
To look at the concentration of urine and plasma in the KCNE1 ko mouse 1. Anaesthetise 2. Cannulate jugular vein 3. Collect urine 4. Blood sample 5. BP- used to ensure the mouse is still out of it 6. Heated pad to ensure temp maintained - KO had no effect on GFR or plasma Na/Cl/fluid - plasma glucose conc was different - looked at the FE% = rate of filtration/ rate of absorption 100= all filtered WT- all absorbed non secreted KO- struggle to absorb Na/Cl/fluid no problem with glucose bc further down proximal tubule it can be absorbed Results repeatable in the Sheffield lab- struggle with glucose as it plays role in late proximal tubule Shows the KO E1 has an effect on tubular function in the nephron
37
Is Q1 regulating E1?
1. Used chromanol 293B inhibitor of Q1 - if E1 regulates Q1 then if you block Q1 in an E1 ko there should be no difference -fe% Na shift - same for Cl and water E1 regulates a chromanol sensitive k channel 2. Patch clamp technique - extracted Q1/E1 k currents- looks voltage dependent When actually look at it it's not voltage dependent
38
Looking at the KCNE1/2 function in gastric function from acid secretion of parietal cells
Ammonium pulse technique - ammonia in joins with H and causes an alkylation - ammonia moved out of cell and H released causing an acidification Look at cells ability to recover - wt= fast recovery -ko= no recovery, wait for new H to come in for exchange, decrease in PH Parietal cell Ko- decrease H, increase PH, alkylation, can't secrete acid so can't digest food in stomach 2. Histamine stimulated- superimposed on top of each other in absence of Na WT= recover HETS= some acid secretion HOM= no acid secretion from acid load- no K secretion as E2 isn't there to regulate Q1

BMS346 (5 decks)

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