EP Flashcards

1
Q

Why are epithelial cells polarised?

A

They have different channels on the apical and basolateral membranes so they can allow net transport across. (Transcellular)

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2
Q

Two different transports from one side of epithelial cells to the other?

A

Transcellular- Net transport through the cells.

Paracellular- Transport between the cells.

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3
Q

What determines the direction of ion movement through epithelia cells?

A

Driving forces- electrochemical (Both charge and concentration)

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4
Q

Tight vs leaky epithelia differences why?

A

Resistance much higher in tight ( greater than 2000 ohm cm2 vs less than 200). The difference is the paracellular resistance not transcellular.
The flux is therefore much greater in leaky and due to the free movement the Vte is 0mv, whereas tight is around 50mv.

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5
Q

Resistance of Tight epithelia?

A

Greater than 2000 ohm cm2. Tight junctions between epithelial cells.

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6
Q

Resistance of Leaky epithelia?

A

Less than 200 ohm cm2

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7
Q

Tight epithelia example?

A

Stomach, Frog skin, distal tubule kidney

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8
Q

Leaky epithelia example?

A

Proximal tubule, gall bladder, small intestine, choroid plexus

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9
Q

What is transepithelial Potential?

A

Vte- difference between one side of the epithelia voltage and the other side- e.g. the potential difference between voltage on the apical and basolateral side of epithelia.

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10
Q

How can the transepithelial potential be measured?

A

Two electrode voltage clamp- An electrode is placed either side of the epithelial cell.
One side is clamped to a known voltage e.g. 0mv, and the resultant other side is measured. E.g. If clamped side is zero but measured side is 40 the Vte is 40 or -40 (depending on which direction taken)

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11
Q

In the patch technique why if one side is clamped to a certain voltage does the other side change?

A

The driving force is changed. Think of an IV curve, depending on what the voltage is, the current through the channel varies (Driving force and some voltage dependant channels). This varying current of ions through will vary the intracellular concentrations and therefore the driving force over the adjacent membrane, so changing the voltage there.

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12
Q

What is Vrev?

A

When net current through a channel is zero. Equilibrium. On an IV curve, the voltage at which the current is zero (crosses the X axis).

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13
Q

What is current?

A

a directional flow of ions through a channel. measured in Amps. Usually uA.

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14
Q

What is voltage?

A

The potential difference in charge generated as a result of movement of ions (current) Measured in Volts, usually mV

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15
Q

What is transepithelial resistance?

A

Rte- resistance of the epithelia e.g. how easy or hard it is for ions to flow from one side of a membrane to another. E.g. Through the tight junctions paraceullar between epithelial cells. if lots of gap junctions resistance is lower.

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16
Q

Why does paracellular resitance vary?

A

Occludin/ claudin for tight junctions between epithelial cells. Tight junctions determine the paracellular resistance, and therefore largely the transepithelial resistance. Tight junctions may also serve as leaky pathways by forming selective channels for small cations, anions, or water. Tight junctions are present only in vertebrates. The corresponding junctions that occur in invertebrates are septate junctions.

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17
Q

Equation to work out resistance of an epithelia?

A

Rte=Change in voltage/ Current injected (Ussing chamber)

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18
Q

Ohms law?

A

V=IR Voltage=Current x Resistance

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19
Q

If there is a high resistance for a given current the Vte is high or low?

A

High. Around 50mv
Tight junction- less leaking of current across after addition one side. Therefore, there is a high voltage difference across both sides (transepitethial potential).

Whereas, if leaky, the current is placed one side and leaks across the membrane dispursing the current (large flux) and leaving no difference between the voltages either side (no transepithelial potential- around zero)

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20
Q

Paracellular or transcellular determines tightness of a membrane? Resistance?

A

Paracellular, transcellular resistance often the same, around 7000ohms

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21
Q

How is Vte generated?

A

The net movement of ions across the apical and basolateral membranes. Depends on which way the ions move, their charge e.g. cl- or K+, and how many move per ‘cycle’

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22
Q

Model stages used for experiments?

A
  1. Fresh tissue/cells- e.g. sharks fin, isolate and if needed enzyme used to break it down.
  2. cultured cells- Used a few days after taken from patient or frozen e.g. HeLa cells.
  3. Whole animal data- WT, KD, KO, Pharmacological agent, overexpression data
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23
Q

Method to test protein is there?

A

PCR is mRNA there? Western blot from gel- for protein

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24
Q

Method to test protein location?

A

Immunostaining- AB to and secondary with fluorescent marker.

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25
Q

Method to test channel flux?

A

Radioactive ion one side e.g NaI (iodine) can measure the amount of net transport by its movement.

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26
Q

Patch clamp measures?

A

Single or whole cell conductance/ current

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27
Q

Two electrode voltage clamp?

A

One clamps potential the other records the generated potential.

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28
Q

What is a short circuit current?

A

Net ion flux across the membrane (so individual currents added).
Often small as there is not a lot of net transepithelial movement

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29
Q

Microelectrode measures?

A

measures potential difference so can create IV curves etc or the single channel recordings.

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30
Q

How can one use a microelectrode to predict whether ion channels are selective for a certain ion?

A
IC potential (Vm) (Current vs Voltage)
Calculate the nernst potential for two ions e.g. K and Na. Clamp the extracellular a potential and then measure the intracellular Vm. If this is near say K nernst, likely to be K channels open at this clamped potential. But confirm by changing the extracellular potential again. This therefore changes the nernst potentials for the ions and if the Vm follows the K nernst, it is likely to be selective to this. Can plug these into an equation to see selectivity ratio using Vrevs.
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31
Q

How are pharmacological inhibitors used for cell channel studies?

A

Can measure whole cell conductance and then use an inhibitor of certain channels and see if the voltage inside the cell changes.
E.g. add amiloride and the potential moves closer to the nernst for K instead of Na e.g. -40 to -60mv, therefore Na through ENaC was contributing to the current.

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32
Q

Amiloride blocks..

A

ENaC channels (Sodium)

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33
Q

Barium blocks..

A

K channels

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34
Q

Current is zero on an IV curve when?

A

The channel is at its Vrev (reversal potential) where there is no net ion flow, or when a channel is closed.

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35
Q

PCR tells us what info?

A

mRNA presence

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36
Q

Immunostaining tells us what info?

A

protein location

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37
Q

Flux radioactive compounds and electrophysiology tells us what info? Experimental evidence.

A

Ussing & Zerahn, 1951
channel transport function- Put Na24 in chamber one (extracellular- krebs solution only in chamber2) Na will transport across the epithelium to chamber 2. The flux from 1 to 2 is measured. The Na24 is switched to in chamber 2 and the flux 2-1 is measured. Flux(1-2)-Flux(2-1) is used to calculate the Net transport of Na (JNETNa) per unit time.

Based on JNETNa per unit time the expected current was calculated. Use Ussing chamber to work out actual Isc (equations). JNET= Isc therefore the movemrnt of Na is all active (Na/k ATPase). There was a little Na outflux leak. Follows Nernst for Na.

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38
Q

What drug stimulates CFTR?

A

Lubisterone, IBMX/Forskolin

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39
Q

If JNET=0?

A

Passive transport only. Using the radioactive Na e.g. Influx=outflux so no net movement.

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40
Q

if JNET not equal zero?

A

There is a difference between influx and outflux of ions across an epithelial (Radioactive Na e.g.) therefore there must be an active element reabsorbing/secreting the ion.
If =zero likely leaky membrane.

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41
Q

All active components if JNET=?

A

ISc- if it equals the short circuit current calculated in the ussing chamber, these active ion channels make up all of the net transport across the epithelium.

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42
Q

If influx into cells from lumen is greater than efflux, the ion is being..?

A

Reabsorbed, if efflux is more- secretion (think kidney- out of tubule lumen into blood)

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43
Q

Apical vs basolateral?

A

Apical is lumen side- the side facing the ‘tube’ inwards where all the transport is finally decided, e.g. in the airways on the mucous side, and in the GI tract the intestine lumen side, kidney PT lumen side (basolateral-blood side).
For CFTR- CFTR always on the apical (controlling secretion/reabsorption) whereas the Na/K ATPase etc on the basolateral, setting up the concentration gradients.

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44
Q

Aim of the Mall 1999 study?

A

Do cystic fibrosis patients have higher Na conductance in the colonic epithelium, as is known to be in the airways? (CFTR inhibits ENaC|)
(Yes)

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45
Q

Mall 1999 Model to study?

A

Human intestinal membrane- 34 non CF biopsies, and 14 CF. Mounted on an Ussing chamber for studies.

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46
Q

Mall 1999 First experiment finding?

A

The Vte was significantly higher (less negative) and the Rte was significantly higher in CF tissues. Non-CF tissues demonstrated cAMP-dependent Cl− secretion that was absent in biopsies of CF patients. E.g. the Vte was unaffected by IBMX/Fors, but did respond to amiloride.
E.g. CFTR channel disfunction, but EnaC functioning.

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47
Q

Why in Mall 1999 was there a basal negative Vte? How did chemicals affect this?

A

EnaC is reabsorbing Na+ so there is a negative potential on the apical side.
When Amiloride was added the potential went to zero- the Na was no longer being absorbed.
When Forskolin/IBMX was added in the WT the potential returned negative (CAMP induced CFTR channels) but on CF tissues- no decrease (CFTR faulty). even increase as activates K channels (+ out).

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48
Q

What were different chemicals impacts on the amiloride sensitive Isc?

A

Non CF- indomethacin increases Na short circuit currents, whereas IBMX/Fors inhbits (CFTR inhibits the channel)

CF-no impact by either chemical but under basal conditions the short circuit current was increased from 6 to 20 (lack of CFTR-dependent regulation of ENaC).

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49
Q

What is JNET?

A

The net transport of ions

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50
Q

Mall 1999 conclusion?

A

Amiloride-sensitive Na+ currents are inhibited by activation of CFTR (IBMX/Fors) only in non-CF but not in CF tissues.

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51
Q

What is indomethacin? Why was it used in the Mall 1999 study?

A

a non-steroidal anti-inflammatory agent (NSAID) that inhibits CYCLOOXYGENASE, which is necessary for the formation of PROSTAGLANDINS. PGE2→CAMP induced CL- secretion, therefore it inhibits CFTR.

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52
Q

Mall 1998 experiment used and found?

A

Nasal cells- easy to access, scrape out the nose. Found that Na reabsorption causes the depolarisation but the addition of amiloride hyperpolarises the cell (from -40 to -60mv) away from the nernst for Na.Therefore showing this channel is contributing to the voltage.

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53
Q

Before 1995 experiment to isolate the ENac protein?

A

Found this very hard. Earlier experiments tried to isolate the protein using an Amiloride affinity column, but needed more Enac than in Native cells to extract the sequence.

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54
Q

When/how was Enac finally sequenced?

A

1995 Canessa-

  1. Took mRNA from the colons of salt depleted rats (induces ENac expression by aldersterone).
  2. CDNA of this into an expression vector.
  3. Chopped up the DNA into 10 groups, cultured to divide.
  4. Inject the pool back into the Xenopus oocyte.
  5. See which of the split mRNAs gave a functional amiloride sensitive Na current. “functional expression”
  6. Further purified this functional pool until purified EnaC.
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55
Q

1995 Canessa- As purified the fraction expected to see increased currents but didnt? Why?

A

Because ENaC is not just one subunit, and as they were purifying were just getting the alpha subunit, which luckily still formed a functional channel or the protein would not have been isolated. But the addition of Y and B made the full 1800nA current. (if just two around 50-80nA).

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56
Q

What other Na channels bar ENaC?

A

The Loop diuretic sensitive NKCC1 cotransporter (2 in kidney) (Barters)
Thiazide sensitive NaCl cotransporter. (Gitelman’s syndrome)

Na+/H+ exchangers
Na+/HCO3- cotransporters

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57
Q

How does the ussing chamber work?

A

Two chambers with the membrane between. One side current is injected which changes the transepithelial potential. The resultant voltage changes are observed (if high resistance epithelia large Vte seen (if low resistance leaks across and disserpated). Resistance and short circuit current can be calculated.

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58
Q

Why do you have to be careful with overexpression data?

A

have to be careful with as limiting factor could be another protein regulating etc, e.g. if upregulatate CFTR without all the other regulating proteins will it work as well? but if then upregulate those proteins- they could be non-specific and have other functions.

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59
Q

Transepithelial resistance equation?

A

Rte=change in voltage/ current injected

Rte=AV/I

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60
Q

Equation for short circuit current?

A

Isc=Vte/Rte

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61
Q

Vte trace?

A

Vte is the top line, the change in Vte is the difference between top and bottom line (maybe?)

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62
Q

If there is a negative Vte?

A

More cations moving in this direction than anions

e. g. if vte negative (0 to -40) there will be either anions secreted or cations being reabsorbed, or both.
e. g. CFTR and ENaC.

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63
Q

Frog skin is a model for?

A

renal collecting duct, distal colon, salivary gland, sweat duct, airway surface epithelium

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64
Q

Why use frog skin?

A

Oocytes- dissect off. Same mechanisms as humans, robust and easy to use.

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65
Q

Another name for absorptive cells in the small intestine?

A

enterocytes

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66
Q

Cellular/molecular structure of the upper airways?

A

Epithelial cells wth cilia.
The periciliary layer of liquid (PCL).
The mucous layer. (With PCL=Airway surface liquid layer ASL).

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67
Q

function of the components of the upper airways?

A

Epithelial cells with cilia- these secrete ions to maintain the PCL height for optimal beating of the mucous.
PCL: height controls beating ability of cilia- too low and the cilia will bend in on themselves, but too high and the cilia cannot beat the mucous back up to be swallowed (HCL stomach).
Mucous: Traps pathogens so they do not get to lungs

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68
Q

Issue with PCL as you go up the respiratory tract?

A

In proximal- alevolar and lots of branching so has a higher surface area to cover of 2m2, whereas as rise converge to one oesophagus where the SA distally is only 50cm2. This means that height of the liquid layer will increase as rises as it has less SA to cover. This is an issue.

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69
Q

Control of the ASL height methods?

A

Active or Passive control.
Passive: Mucous part acts as a reservoir and can passively move into the mucous layer.
Active: Net transport of ions into the PCL controls the osmololity so water will follow/ be reabsorbed.

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70
Q

In vitro the ASL settles at what height? How?

A

2001 study:
7um- if artifically add water to Cultured human airway epithelial cells to make the layer rise (25um) it will reduce and stabilise at 7um in just over a day.

If also measure Vte, follows the same pattern- the transepithelial potential changes to rescue the height, e.g. upregulation of transporters- reabsorb ions so water decreases so height decreases.

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71
Q

What height is the ASL at in vivo?

A

around 14um- which can be given in vitro if the layer is mechnically stimulated like the rhythm of breathing.

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72
Q

What inhibits the Vte change as the ASL layer is trying to be rescued after artificially increasing it?

A

2001 study:
Bumetanide blocked cl- secretion:
- At 0hrs 18% of he Vte is blocked
- At 48hrs 55% of the Vte is blocked (much bigger action at rest)

Amiloride blocks Na absorption:

  • At 0hrs 65% inhibition of Vte
  • At 48 hrs- 30%

At 0hrs- reabsorbing more and secreting less- osmolality decrease, water reabsorbed, layer decreases.

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73
Q

NKCC1 inhibited by?

A

bumetanide and Furosemide

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74
Q

bumetanide inhibits?

A

NKCC1 on basolateral airway epithelial cells.

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75
Q

ASL height determined by which solutes?

A

Tha balance between Cl- secretion (NKCC1 Baso and apicalCFTR) and Na absorption (apical ENaC).

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76
Q

What are the 5 key channels on the airway epithelial cell?

A

Apical: ENaC and CFTR.
Basolateral: Na/K (3Na out: 2K in)- sets the driving force for Na in through ENac and NKCC1 (with cl). Now lots of K inside needs a K channel to leak out.

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77
Q

Virus that affects the balance in the respiratory tract?

A

respiratory syncytial virus. Nasal conjestion (runny nose), bronchiolitis (20% of hospital administrations for children) and Pneumonia in adults.

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78
Q

How can the effect of RSV be measured? Result?

A

The Enac current is measured before and after the RSV ( by addition of amiloride to see difference in overall voltage change)
Without RSV the voltage change after amiloride is the same 1 hour later (the Enac current therefore is the same). But with the addition of RSV aftert he first measurement, the second had an overall rise in voltage but the same voltage after amiloride addition- meaning that the amount amiloride reduced the voltage was less as there was less Enac to inibit but it inhibited it to the same final amount. short circuit current was reduced.
THEREFORE RSV inhibits ENaC.

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79
Q

What does the inhibition of ENaC by RSV mean?

A

If block EnaC, less Na is absorbed so more in the airways- the excess fluid is lost through the nose- runny nose.

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80
Q

Through what mechanism does the RSV act?

A

RSV activates PKC which inhibits ENaC. It binds to glycolipids also.

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81
Q

How do we know how the RSV works?

A

NA – neuoaminidase, inhibits binding to glycoproteins (no impact- Enac Isc same as RSV alone)
BIM – PKC inhibitor
PPMP – inhibits binding to glycolipids
both inhibited the action of RSV so stopped the virus functioning.

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82
Q

Other viruses use which methods to influence the PCL?

A

Influenza- PKC and glycoproteins

Parainfluenza- Glycolipids, ATP release- inhibits ENaC.

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83
Q

Influenza kills how many people a year?

A

36,000

84
Q

Viron particle structure?

A

M1 matrix protein
Haemagglutinin – binds to sialic acid residues-activates PKC & transient inhibition ENaC.
M2-forms an acid activated, amantadine inhibited H+ channel, inserted into apical membrane host cell during infection

85
Q

Is M2 involved in the long term inhibtion of EnaC experiment?

A

IV curve patch clamp of EnaC with and without M2- M2 takes the current to zero.
Po? Single channel conductance- open probability
decreased with M2.
N? Western blot- decreased Enac a and B at the cell surface as overexpressed M2

86
Q

M2 with Liddles?

A

The effect of M2 is reduced if the ENaC has a liddles mutation. Liddles mutations makes the ENaC unable to be endocytosed out of the membrane (GOF)

87
Q

ROS and M2?

A

Where M2-RFP is overexpressed high expression of ROS- coexpressed. Control of RFP only is done to ensure doesnt upregulate ROS.
If use GSH which is an antioxidant (mops up ROS) some function of ENaC is restored.

88
Q

What is PHA?

A

pseudohypoaldosteronism. ENaC LOF.

89
Q

Mutations involved in PHA1?

A

Dominant: mineralocorticoid receptor mutation. Kindey form.
Recessive: All subunits ENac mutation. Systemic- mutiple organs effected expecially respiritory tract which is prone to infection.

90
Q

PHA1 symptoms and explaination: (Kidney dont need to learn?)

A

Salt wasting: LOF ENaC- less Na reabsorbed- more lost.
Hypotension: Water follows the Na, if less reabsorbed, less water reabsorbed- EXV down, BP down
Hyperkalaemia:
Metabolic acidosis:
High renin & aldosterone: trying to compensate- put lots of AQP2 into membrane to reabsorb more water from collecting duct.

91
Q

PHA1 systemic type symptoms? Causes?

A

ENaC LOF- less absorbed Na, so increased osmolality of PCL- increase water in- runny nose.
Normally around 20mg out nose they have 45mg .
Vte- control -20mv, PHA1=-7mv (less negative potential, less + inside and - outside (PCL)
Amiloride only reduced the Vte by around 5% compared to 58% in control. (LOF EnaC)
Increased PCL- cilia cant beat efficiently- increased risk of infection.

92
Q

epidemiology of cystic fibrosis?

A

Cystic fibrosis (CF) is the most common fatal genetic disorder of caucasians. 1/25 carriers, 1/2500 CF.

93
Q

What impact can CF have on the pancreas?

A

Pancreatic Insufficiency. In the pancreas, occlusion of ducts (and lack of HCO3−) prevents pancreatic enzymes from reaching the lumen of the intestine and may lead to premature activation of digestive enzymes inside the pancreas causing pancreatitis that may also cause diabetes mellitus. Approximately 80–85% of CF patients have pancreatic insufficiency, which leads to decreased availability of both digestive enzymes, so results in babies not gaining weight.

94
Q

CFTR not only tranports Cl- through but also?

A

HCO hydrogen bicarbonate

95
Q

Cystic fibrosis symptoms?

A

Struggle to put on weight, increased sweat Cl- (Guthry test above 60mmol), male infertility (vans deferens missing), Airway disease.

96
Q

How to measure height of ASL?

A

Height: The diffusion of FITC-dextran (flourescent) dissolved in the ASL of human bronchial epithelia grown at an air-liquid interface was used to visualise and measure from z-image confocal stacks, the height of the ASL.

97
Q

How to measure ASL viscosity?

A

Photobleaching using a laser was done which reduced the fluorescence stain. The rescue of the fluorescence was measured. In the Cystic fibrosis patients mainly under 10% of the fluorescence was restored in due to the thick mucus. Diffusion was measured by fluorescence recovery after photobleaching, using a perfluorocarbon immersion lens and confocal fluorescence detection.
Standard linear graphs were created plotting viscosity against the time that half or ¼ of the fluorescence was returned. I.e. If higher viscosity (thick), the longer the time took to recover.

98
Q

(Leaky) epithelia capable of secreting NaCl rich fluid include:

A

exocrine gland acini • sweat gland coil
small intestine • upper airway
choroid plexus
and Shark rectal gland

99
Q

Why is shark rectal gland used in research?

A

In the US where first used was very easy to access.
They have a large amount of secretion so easy to measure.
Very robust- they last for hours in vitro in an organ bath, compared to mammalian tissue which dies quickly.

100
Q

Furosemide inhbits what?

A

NKCC1- brings Ecl to equilibrium (passive) so stops the active component of Cl secretion.

101
Q

Ouabain inhibits what?

A

Na/K ATPase- which stops the driving force for NKCC1 and EnaC and CFTR, so it stopped Cl- secretion.

102
Q

how can one tell there is an active component involved in Cl- secretion?

A

If know the intracellular and extraceullular Cl concentrations can work out the nernst for Cl-. Got 17mM or 28mM (depending on over which membrane), yet the membrane potential was -70mv, so there must be a compenent that causes the accumulation of Cl- inside the cell. ABOVE ELECTROCHEMICAL EQUILIBRIUM

103
Q

What is the advantage of keeping Cl- above the electrochemical equilibrium inside the epithelial cell?

A

so only need to open a channel and cl- rushes out through CFTR, creates a passive driving force.

104
Q

Cl- secretion is …. activated?

A

CAMP

105
Q

CFTR gene makes what?

A

Cystic fibrosis gene is CFTR Cl channel.

106
Q

CFTR structure?

A

On apical membrane, 12TMD- 1 subunit forms full channel but lots of other proteins interract.
Split up into 2 TMD’s of 6.
2 nucleotide binding domains (NBD1 and 2)
R regulation site that can be P by PKA- CAMP activation.

107
Q

CF mutations?

A

Over 1200 all over the protein, but clusters at NBD1/2 e.g. AF508 in NBD1.
I: Null production: mRNA down, unstable breakdown
II: Null trafficking: misfolded so degraded (QC) AF508
III: Regulation-made and gets to the membrane but Po down due to regulation changes.
IV: Conduction- Po reduced.
V: Partial reduction mRNA- not enough made N down.
VI: High turnover- endocytosed quickly and degraded.

108
Q

Need what percentage of CFTR for normal function?

A

15-20%

109
Q

Significance of the variety of mutations for CF?

A

Different severities (class I-IIIaround 100mmol, IV-V- 70-80mmol), different drugs work (personalised to type),

110
Q

Clinical threshold for CF?

A

Above 60mmol salt in sweat.

40-60mmol inconclusive.

111
Q

What is the most common CF mutation?

A

Delta F508, upto 90% of CFs are, its a pheylalanine deletion which causes the misfolding of the protein so its targetted for degredation. If the protein gets to the surface though it can have some function. (membrane trafficking journal club)

112
Q

Model cell for CFTR? WHy?

A

Rat colonic crypt cells- cl- is secreted out into the faeces etc to control the water content in the faeces.

113
Q

How does the rat colonic crypt cell CFTR function?

A

Na/K ATPase on basolateral creates driving force for Na in through NKCC1. 2 CL- in cell and secreted through the apical CFTR into faeces. Also an apical K secretion channel, which further causes a driving force for Cl- secretion due to charge balance.
Na paracellular transport.

114
Q

In rat colonic crypt cell what activates the different channels?

A

Prostaglandin PGE2 increases CAMP which activates CFTR.
Ach stimulates Ca which activates the K channels-secretion. (driving force for CFTR then). Possibly activates the Na/K ATPase but not known.

115
Q

Pharmacological compounds that stimulate CAMP? (CFTR link)

A

Indomethacin inhibits Prostaglandin production so reduces CAMP production.
IBMX inhibits phosphodiesterase (breakdown of CAMP) so CAMP increases.
Forskolin- activates adenylyl cyclase- increase CAMP

116
Q

WHat does CCH do?

A

Carbachol CCH- Ach R activation- increases Ca- increases K secretion- increases Cl secretion
(in rat colonic crypt).
Can see on graph (lecture) CCH causes the Vm to reduce.

117
Q

Pharmacological compounds difference on CF patients?

A

No change in Vm (Cl- secretion) with CCH, and Indomethacin which in control inhibited the Cl- secretion increase, had no impact as no Cl- was being secreted. IBMX/ Forskolin also had no impact (where in control restored cl- secretion)

118
Q

Why would IMBX/ Forskolin rescue the action of Indomethacin?

A

Because IBMX/Forskolin act further downstream- Indomethacin inhibits Prostaglandins which cause the increase in CAMP (so reduce CAMP levels) but Forskolin activates Adenylyl cylase so increases CAMP directly and IBMX reduces the breakdown keeping levels high, so they act later byapssing the action of Indomethacin.

119
Q

CF and colon?

A

10% of CF babies have meconium ileus which needs surgery in days. Its the thickening of meconium (the substance creating the first faeces of an infant), creating a blockage. (Less watery more thick)

120
Q

relationship between CFTR and ENaC?

A

CFTR inhibits ENaC. So in CF patients ENaC is further upregulated so this further emphasises the low osmolality of the liquid layer.

121
Q

Difference in the alveolar epithelial cell model?(3)

A
  • No NKCC1 on the basolateral side. instead has Cl, K cotransporters.
  • And works the opposite way- Cl- sits lower than equilibrium so if the channel is opened Cl- will be absorbed and Na.
  • ENaC and CFTR work together- both active or inactive.
122
Q

CF symptom in the alveolar lower airways?

A

As Cl- inwards through CFTR, if CF too much Cl- outside. Water follows. Can get pulmonary oedema.

123
Q

Why do CF patients have salty sweat?

A

Different cell model, Cl- secreted from one set of cells (not CFTR other cl- channels) and reabsorbed through others. But the CFTR in those that reabsorb not functioning.

124
Q

Viscous cycle for CF patients in upper airways?

A

Mucous build up (viscous)- bateria-infection-inflammation-tissue damage- decrease in airway function.

125
Q

Word for thick sticky when describing mucous?

A

Viscous

126
Q

What are Mycolytics?

A

E.g. Pulmozyme They break apart the mucin in mucous so its easier to cough up for the patient.
Mucin= heavily glycosylated protein that forms the gel in mucous. These hydrolyze the DNA present in mucus of cystic fibrosis patients and reduces viscosity in the lungs, promoting improved clearance of secretions CHECK.

127
Q

Advatages of Mycolytics?

A

Usually v good, suggested the life expectancy increase of 5.3% from 2002-2003
a large randomised placebo controlled trial was set up with nearly 1,000 adults and children with CF and reduced lung function who were treated once or twice daily for 24 weeks.This showed reductions in respiratory exacerbations of 28% and 37%, respectively. And children on saw fewer microbe cultures growing in their lungs.

128
Q

Disadvantages of Mycolytics?

A

Treat symptom not disease.
The major component of mucus in CF is not mucin derived from mucus producing cells but pus. So degradation of Mucin may not be the most effective treatment.
30% of patients appear not to respond to pulmozyme. Median decrease in sputum (mucus and saliva coughed up) elasticity was 32% in patients the treatment that respond to the treatment compared to 5%, which can mean that they cough up in parts inctead of all together. Newer agents targeting other components of CF mucus, such as filamentous actin, are currently in development. Preserve viscoelasticity.
(CHECK)

129
Q

Nebulised hypertonic saline impact on cystic fibrosis?

A

Breathe in Nebulised hypertonic saline sets up an osmotic gradient to increase PCL height. Australians use as Pulmozyme not licenced. Australian study found significant 3% increase in FEV1 in CF patients after use, but no impact on pulmonary function after 48 weeks.

130
Q

What is FEV1?

A

The maximal amount of air you can forcefully exhale in one second. Normal range is 80% of predicted for average height age etc.

131
Q

CF patients treatment to reduce inflammation?

A

Oral antibiotics, nebulised antibiotics, and steroids.

132
Q

Two Vertex drugs?

A

VTX-770- Ivakaftor- potentiator licenced for G55ID in UK. (potentiates opening)
VTX-809- Corrector (corrects folding)

133
Q

Other CF that Ivakaftor is for?

A
G55ID- Glycine to aspartate. Mutation which abolishes the ATP dependent gating so has a 100 fold lower Po- regulation class problem.
1-3% pop have.
134
Q

How could vertext see if drugs had impacts on the G55ID CF CFTR?

A

Used cell- based fluorescence- When cell voltage changes, the fluorescence changed so could see which drugs opened the CFTR channels.
228,000 chemicals tested.

135
Q

How could vertext see if drugs had impacts on the AF508 CF CFTR?

A

Look for a compound that would drive the CFTR to the membrane. Do a cell based immunoblot assay (western blot) to see which shifted the band: b to C at the PM.
164,000 tested chemicals.

136
Q

2009 Vertex study for G55ID animal experiment?

A

On Fisher rat thyroid.
WT: Forskolin increased epithelial cell voltage from 10mv to 50mv. (as Cl- out more +)
G55ID: 2mv to only 5mv.
G55ID+VTX770 to 20mv.
Current through channel shows simialr results.

137
Q

2009 Vertex study for G55ID human cells first experiment?

A

Test on Human bronchiole epithelia (HBE) Isc
WT FSK response = 56 ± 6 µA/cm2
G551D/F508 +FSK response = 2.9 ± 0.5 µA/cm2
~5% of WT
G551D/F508 + VX-770 FSK response = 27 ± 2 µA/cm2
~48% of WT same as carrier- asymptomatic
Clinically suggestions 15% sufficient to alleviate symptoms in CF patients

138
Q

2009 Vertex study for G55ID human cells second and third experiment?

A

ALS height: VIP stimulates CAMP, if increase height to 100um and see where it stabilises at, from around 40 to 30um (WT around 14)
ciliary beat frequency: Mutant 2Hz on own, add VIP and VTX-770 to 15hz, where WT around 16hz.

139
Q

2009 Vertex study for G55ID clinical trial experiment?

A

Ranom double bind placebo Clinical trial 161 patients with at least 1 G55ID -mutation -measured FEV1 change over 48weeks.
Vtx770- improvement: over a 10% increase in FEV1, whereas placebo 2% worsening.
Sweat dropped below 60mmol. (100 to 50)
Also, 67% of patients on drug not having any events , compared to 41% in placebo group.
(events being pulomonary excacebation, infection, weight loss, increased coughing- often admitted to hospital- NHS money saving)
IN VIVO PATIENT IMPROVEMENTS.

140
Q

2009 Vertex study for G55ID clinical trial experiment also measured..?

A

Transepithelial potential in nasal epithelial cells, by squirting in Cl- free saline liquid, to create a driving force for Cl- secretion and add a activator of CFTR and measure the change in voltage.
There was a negative shift as Cl- was being secreted, saw an improvement.

141
Q

Vertex 2011 study VX-809 for F508 animal study experiment?

A

Fisher rat tyroid tissue.
Measured a ratio of immature (band b) to band C (mature) from western blots. As concentration (until plateau) increased the ratio increased. And short circuit function also increased.
Corrects the misfolding so allows the trafficking of some of the CFTR.

142
Q

Vertex 2011 study VX-809 for F508 first experiment on human cells?

A

HEK cells- pulse chase experiment.
Add radioactive cysteine/methionine 35S and wait for it to incorporate into the CFTR protein, nd cells left for different durations.
Chase follows the processing of the CFTR- some glycosylated (mature) some not (immature). Ratio depends on time left etc.

Results:
First can see it incorporated into immature then shifts as matures at 60mins, then gets degraded around180mins.
F508= just disappears after 60mins, doesnt mature.
F508+ Vx-809- mature appears and remains similar time but less made (fainter).

143
Q

Vertex 2011 study VX-809 for F508 second experiment on human cells?

A

HBE tissue from patients -/- F508

Short circuit current increased with Vx-809 after CAMP agonist addition. Increases with dose also.

144
Q

Vertex 2011 study VX-809 for F508 clinical trial experiment?

A

Randomised double blind clinical trial-19 placebo, 19 on Vx-809 in different concentrrations for 28days.
FEV1: stayed the same or decreased even.
The sweat Cl- level decreased by 8mmol- yet VX-770 was by -55mmol! Combination therapy?

145
Q

Vx-809/ Ivacafter called? Drawbacks? benefits?

A

Orkambi

  • Very expensive
  • Sweat Cl- down 13mmol, higher but not like VX-770 alone for G77ID.

But 6% increase in FEV1- with 600/250mg dose, but monotherapy decreased FEV1.

146
Q

Phase 3 clinical trial of Orkambi VX-770/VX-809 combo for F508 results?

A

Trial on 1108 people over 12 years 2015.
FEV1 increased around 3%, with the greatest percentage of people having an increase of over 5%, some over 10%, but some worsening. (would normally get worse over time).
60(placebo) to 80% of patients not having an event over a 45 week period.

147
Q

Cost of Ivacafter? Vs Orkambi? Which licenced why?

A

I: £189,000 per patient per year £200 thousand.
Licenced in the UK for G55ID.
Decreases hospitalisation so saves money here, and improves the symptoms for the patient.
(FEV1-10% increase, salt -55mmol (below 60), %of patients event free from 41% to 67% 48 weeks)

O: £104,000 but not licenced, the patient will still need current medication, and still substatial hospitalisation, less impacts on the patient.
(-12mmol drop cl- sweat, 3% FEV1 increase, pulomary exacerbation 40% (45% hospitalised, 60% IV AB) to 20% (20% hospitalised and 30% IV).

148
Q

Initial problem with CFTR gene therapy?

A

1990 first showed promise, but due to high cell turnover rate need to keep adding- aerosol with adenovirus with the CF gene in to make CFTR in the body.

149
Q

2015 development of gene therapy for CF? Method?

A

liposome complexes contain the CF gene
Clinical trial 140 patients, (62- placebo- nebulised saline- would be ideal to give liposomes with scrambled CFTR but not ethical incase make another protein and not healthy already)
28day intervals of 9 doses.

150
Q

2015 development of gene therapy for CF? Results?

A

Stabilised the FEV1 (where placebo decreased over time ). Best result 20% improvement.
Questions whether the liposomes affected the results at all- whether this was the best delivery method, but without placebo with liposomes alone can’t say. More research needed.

151
Q

What percentage of CF patients have only one mutation?

A

1-2 northern europe.

152
Q

Atypical CF means?

A

Mild symptoms (40-60mmol sweat)
1 or NO CFTR mutations
Carriers typically no symptoms, so odd to see symptoms, maybe other mutations? ENaC GOF?

153
Q

Experiment on atypical CF patients to investigate the genes involved?

A
screening of 31 patients genes- 
SCNN1A (alpha subunit)- 11 mutations. 
SCNN1B- (Beta)- 7 mutations.
SCNN1G- (Gamma)- 8 mutations. 
on 45 patients-Same as above but also some other mutations found- some LOF??
154
Q

Nasal studies on Atypical CF patients- at rest?n What do the three experiments tell us?

A

Electrodes up nose measure the Vte.

  1. The Vte measurements for the E528E (mild Cf polymorphism)/ W493R enac mutation or AF508 =/1 W493R have intermediate VTEs betwen normal and classical CFTR. Hyperpolarised- cell more negative.
  2. The Cl- secretion levels are close to the WT after adding cl- free solution to promote. PD decreases as secretes Cl- (-16 WT, atypical -20/-15, CF= +0.1 )
  3. amiloride sensitive current is upregulated- but unlike Classical CF which is due to loss of inhibition by CFTR, the atypical still had CFTR working so is just GOF of Enac. (6.5mv change to around 20mv).
155
Q

Nasal studies on Atypical CF patients- at rest? With additional low Cl- solution.

A
  1. WT, 2. classical CF 3.E528E/w493R 4. f508/w493RIf add low cl- solution (drive Cl secretion). Meausred the PD change
  2. -16
  3. 0.08- no cl secretion cftr dysfunction
  4. -20mv CFTR working as WT
  5. -15 CTR working as WT
156
Q

Nasal studies on Atypical CF patients- at rest? Change in amiloride sensitive EnaC current?

A
  1. WT, 2. classical CF 3.E528E/w493R 4. f508/w493RIf Measure amiloride sensitive Enac current.
  2. 6.5
  3. 19.4
  4. 16mv GOF
  5. 20mv GOF
    But not just because of lack of inhibition by CFTR like in Typical CF, but GOF.
157
Q

Explaination for atypical CF?

A

GOF Enac- Increased Na absorption from the mucous into the epithelial cell. The epithelial cell will be hyperpolarised, and the osmolailty will be decreased in the mucous, making it viscous.

158
Q

What is the most common mutation for atypical CF?

A

The alpha W493R mutation-Found on the extracellular loop of the alpha subunit. No endocytosed out of membrane in negative feedback loop.

159
Q

If overexpressed alpha W493R? Feedback inhibition?

A

Measured the ENaC current by the inhibition by Amiloride- see change in voltage. Although the current is massively enahnced, the percentage difference is the same from high to low Na in the membrane. Short circuit current higher in low sodium.

160
Q

ENac feedback loop?

A

As Na rushes in through ENaC, ENaC is endocytosed. They are then recycled back to the surface (feedback inhibition). Can see after the initial rise in current

161
Q

If high sodium outside? ENaC response?

A

high Na outside, Na rushes in, Enac channels will endocytose. Negative feedback

162
Q

Cleaved vs uncleaved ENaC?

A

Uncleaved: Near silent, Low Po, Generate smaller ENaC currents

Cleaved: By proteases- Very active, High Po, Generate larger ENaC currents.

163
Q

Current depends on which factors? (equation)

A

I = N.Po.g.(Vm-Ei)

164
Q

Is the Enac GOF to do with cleavage?

A

Cleaved- v active. Already cleaved?
Response to chymotrypsin lost which normally increases current through by cleaving the ENaC.
No activation of near silent channels. (look at graph)
Reduced response to chymotrypsin not due to increased endogenous cleavage before chymotrypsin added, but to another factor. The gating is different on the aW493R.

165
Q

Summary the ENaC GOF 3 factors to consider?

A
  1. sodium feedback inhibition- no percetnage change Current low to high Na- ENaC endocytosed as quickly.
  2. Cleavage-no- just different gating not to do with already being cleaved (more active form)
  3. Sodium self inhibition- Yes missing-HIgh Na reduces the Po of ENaC in WT but prolonged high Po in W493R
166
Q

Soidum self inhibition? ENaC GOF?

A

If high sodium into cells feeds back to reduce the Po of ENaC.
This is missing in the GOF EnaC. Current prolonged instead of a V shape with a decay, U shape with prolonged current at the max. Lasts longer.

167
Q

What is the cause of the GOF of the ENaC W493R?

A

loss of sodium self inhibition- where high Na reduces the Po of ENaC.

168
Q

BV348M ENaC GOF mutation – Change in what?

A

2013 study
Po.
Wt- 0.24- open 24% of the time.
BV348M- 0.33- 33% open- ENaC GOF- more Na into cell- water leaves- PCL height down- mucous more viscous.

Western blot showed no change in number of channels in the PM. Single channel conductance also supported as was open more than closed.

169
Q

BV348M ENaC GOF mutation – How measured the Po?

A

2013 study
From 4uA in WT to 5.5uA.
Measure starting current, then expose ENaC to MTSET binds to cysteins- which stabilisises it in an open state so Po=1 and remeasure the current. The ratio of start current vs current when Po=1, gives the Po %. e.g. 12uA before to 20uA- 60% were open.
(Control used MTSET on a cystein mutant to check didnt change current)

170
Q

Which is the rate limiting subunit of ENaC? If overexpress this? (6)

A

SCNNIb- Beta subunit.
1. Mouse overexpress- ASL height reduced.
2. Mucous clearance decreased.
3. And if took a slice through lungs WT had open tubules but blocked in the overexpression mouse.
4. Post-natal mortalities-by 28days half the mice had died.
5. Intratrachea injection-Bacteria clearance decrased- 3 days to clear in WT, still around half there in mutant.
6. Inflammation increased.
General atypical CF like symptoms.

171
Q

Results of ENaC Beta subunit overexpression mouse.

A

Mucous clearance decreased. And if took a slice through lungs WT had open tubules but blocked in the overexpression mouse.
Post-natal mortalities-by 28days half the mice had died.

172
Q

What is the role of the K channels in epithelial cells?

A

They help maintain the negative resting potential- Pump K out (nernst Ek -90mv.) Hyperpolarised.
Important creates a driving force for Cl- secretion and Na absorption when the channels open (NKCC1).
Also K channels have a role in epithelial cell volume regulation e.g. in response to cell swelling.

173
Q

Three families of K channels?

A
  1. Voltage gated Kv channels
  2. Inwardly rectifying Kir channels
  3. Two pore domain
174
Q

Structure of the voltage gated K channels?

A

4 subunits form one channel.6TMDs with the 4th being the voltage sensor and the 5-6 making up the pore.

175
Q

Structure of the inwardly rectifying Kir channel?

A

4 subunits make one channel- only 2TMD with the pore between.

176
Q

Strucure of the two pore domain K channels?

A

1 Subunit makes the channel- 4 TMDs with each 2 tMDs forming a pore (so x2 pores)

177
Q

Voltage sensitive K channels regulation?

A

KCNQ1-Regulation by KCNE1, E2, E3

Kv subdivision that is activated by Ca e.g. SK4 or BK.

178
Q

Example of location of an inwardly rectifying K channel?

A

Kir1.1 ROMK in kidney

179
Q

Basolateral K channel and CFTR channels on upper airway epithelial cells relationship?

A

K out over basolateral membrane. K channels and CFTR tend to be activated at the same time and increases Cl- secretion as it makes the inside of the cell more negative

180
Q

KCNQ1 gene makes what protein?

A

KVLQT1

181
Q

KCNQ1 inhibited by?

A
Chromanol 293B (blocks only a few K channels- relatively specific, but not only blocking Q1) Can use to show whether there is functional KVLQT1 channels. 
Problem: blocks other channels, so needs backing up with mRNA and protein expression studies.
182
Q

KvLQT1 mRNA found where?

A

Study:

Northern blot showed expression in WT, CF nasal tissue and HBE epithelial cells.

183
Q

KvLQT study ussing chamber results?

A

Controls Cl- secretion, measure this instead. Ussing chamber on nasal epithelium (Vte measure)
As increased concentration of 293B (inhibitor), the Vte shifts closer to zero (therefore cell less negative- less K out, reducing driving force for Cl- secretion), showing that this is contributing to maintaining the negative resting potential.
IMBX/Forskolin- increases CAMP so increases KYLQT1 activity also.

184
Q

KvLQT study current results

A

As 293B conc increases, K Isc decreases (prevent cl- secretion). However there is still a persistant current (doesnt block all K channels), but if add barium (general K channel blocker) current goes to zero- so there are likely other channels besides the KvLQT channel working in nasal epithelial cells.
(done in the presence of amiloride to block ENaC which may interfer with results)

185
Q

293B sensitive Isc CF vs non-CF?

A

Isc before and then after 293B addition to find difference.
Non-CF: Increases from 5 to 25uA/cm2 after IMBX/Forskolin. As CAMP inceases cl- secretion increases and K secretion.
CF: Very little 293B sensitive current before or after cAMP (2uA/cm2)- CFTR disfunctional- reduced secretion, KvLQT1 activity cant see.

Barium sensitive Isc is the same after IBMX/Forskolin for CF but much higher 15uA/cm2 - There is Cl- secretuion driven by these barium sensitive K channels through a different Cl- channel.

186
Q

CF KvLQT1 expression in CF patients?

A

Expression is normal, so the reduced function is due to the dysfunctional CFTR (what measuring) and CAMP maybe to blame?

187
Q

The fact that barium had no difference in K currents ater IBMX/Forskolin means what?

A

That there are likely other K channels that are not sensitive to CAMP, and not sensitive to 293B. These were no different in CF patients.

188
Q

2009 atypical CF paper? Aim and results?

A

Aim: Investigate whether mutations in different subunits of ENaC would cause CF like symptoms.

Findings:
a more than three‐fold significant increase in incidence of several rare ENaC polymorphisms was found in the atypical CF patient group (30% vs. 9% in controls e.g. p.W493R in SCNN1A.)
p.W493R‐SCNN1A polymorphism was even found to result in a four‐fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes and increase nasal amiloride sensitive Na currents.

189
Q

Why wouldn’t the hyperactive SCCN1A give CF like symptoms?

A

A four-fold higher amount of SCNN1A transcripts compared to SCNN1B transcripts were found by transcriptome analysis, so likely only the B is the limiting factor for functional ENaC channels, so the other subunits overexpression don’t impact Amiloride sensitive Na currents.

190
Q

KVNQ1 mutations cause what in the cardivascular system?

A

Mutations in KCNQ1 can lead to dysfunction of the channel and cause the cardiac long QT syndrome LQTS1, which in turn may lead to serious arrhythmias, ventricular fibrillation, and cardiac arrest-
They form the K rectifying current to repolarize the cardiac tissue. Voltage gated channel, and CAMP regulated.

191
Q

KVNQ1 role in the digestive system?

A

In stomach KCNQ1 seems to be primarily located in parietal cells and to have an essential function for gastric secretion. KCNQ1 channels in stomach are, in contrast to intestine and colon, located in the apical membranes of the epithelia. The functional channel complex is most likely an interaction between KCNQ1 and KCNE2, even though the presence of both KCNE1 and KCNE3 has also been suggested in stomach epithelia

192
Q

CF other K channels and CL-?

A

hSK4- Ca activated K channel.
CaCC- Ca acitvated Cl- channel.
Work together to support Cl- secretion in CF patients?

193
Q

hSK4 blocker?

A

Clotrimazole.

194
Q

UTP activates what?

A

UTP- purine receptors-intracellular Ca rise- activates CaCC and hSK4 (Cl- secretion increased thrugh both)

195
Q

UTP impact on Vte?

A

Small hyperpolarising shift in Vte (Cl- secretion increased, lumen gets more negative)

196
Q

UTP impact on CF tissue?

A

Isc: uA/cm2. Enhanced in the CF patients. (more negative- e.g. Cl- secretion) nearly double.
COMPENSATION UPREGULATION.

197
Q

Why do CF mice not have any lung problems?

A

Due to an upregulation in the Ca activated Cl- secretion. (UTP experiment)
In humans not sufficient to replace CFTR.

198
Q

Experiment to show Clotimazole blocks Ca activated Cl- secretion? (no CAMP)

A
  1. UTP increase intracellular calcium, measure the Cl- secretion.
    If add 293B not much effect (CAMP low so not suprising KCNQ1 not active).
    However add Clotrimazole (blocks hsk4 ca activated K channel), the UTP increase current disappears.
199
Q

Experiment to show Clotimazole blocks Ca activated Cl- secretion? (with CAMP)

A

With IMBX/Forskolin and UTP.

UTP increases Ca stimualted Cl- secretion, 293B now inhibits this slightly (KCNq1 is playing a part to drive Cl- secretion through CaCC) Clotrimazole added nowstops- major role.

CF: enhancer of Ca activated Cl secretion. 293B inhibits this a lot (KCNQ1 contributes to driving force for Ca activated secretion instead of through CFTR). Clotimazole completely prevents.

200
Q

Two different paired channels in upper airways? (not enac)

A

KCNQ1 and CFTR both CAMP stimulated.

hSK4 and CaCC both Ca activated (upregulated in CF patients, and can use KCNq1 to drive through CaCC too- unlinked)

201
Q

A third K channel in upper airway cells?

A

On the apical membrane. Bk channel. hypotheisis to drive Cl- secretion also.

202
Q

hSK4 vs BK Ca activated channel difference?

A

SK4 is an intermediate conductance Ca activativated K channel. Fewer K ions through per unit time.
BK channels are large conductance- more per unit time.

203
Q

Evidence of BK channel location?

A

HBE cells.
ATP (same as UTP). Stimulated BK channels.
If block BK channels on apical side (Paxilline) response to ATP is lost and decrease Cl- secretion.
Paxilline on basolateral no effect.Therefore on apical.

204
Q

BK K channel KD?

A

See effect on ATP activated Ca activated Cl-short circuit current. Reduction of Cl- secretion. (control scrambled siRNA no impact).

205
Q

Cilia beat frequency method to measure?

A

HBE cells.
cells grow basolateral down apical up. put on a well plate- either put media either side or just below and at the air liquid interface they make their own ASL. Can change media below and see impact on ASL and cilila beat frequency which can be measured.
If manually add optimum ASl height- control to check disrupted ASL not cilia themselvves.

206
Q

BK and ciliarly beat frequency?

A

When paxilline added CBF reduced from 10Hz to 5hz- predict due to disruption of the ASL by BK channel blocking by Paxilline.

If then add PBS to optimum height- CBF back to control.

BK KD. CBF virtually not existant, and PBS addition rescues again.