Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BMS 109 > EP2 > Flashcards

EP2 Flashcards

1
Q

Permeability to water when there is no osmotic gradient?

A

Diffusional water permeability (Pd).
Continuous movement into/out of the cell.
Influx=efflux. No volume change.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Permeability to water when there is an osmotic gradient?

A

Osmotic water permeability (Pf) e.g. cell in hypertonic solution.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Pf/Pd ratio?

A

Directional water movement/ bidirectional water movement.
If ratio greater than one: There is a water pore present in cell membrane (as directional movement).
e.g. NA/glu cotransporter has water pore properties.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Study on Pd/pf in cells? Vague method and results?

A

1953 mesured Pd/pf in a variety of cells using the cartesian diver balance method.
Greater than 1 (water pore) in all cells tested bar the trout eggs.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How to measure Pd?

A

Cartesian Diver Balance.
Diver balances at a varaible equilibirum in a heavy water (D2O) chamber according to cell weight and pressure of chamber.
As cells exchange D2O for H2O they will pull the diver down in the chamber as they get heavier. The amount of pressure needed to be added into the chamber to keep the diver at a given height is measured. (Add vaccum diver rises).
suction needed proportional to the water diffusion.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

1953 cell study Pd results?

A 
All cells (bar trout) had to keep having suction put into the chamber but less and less over time as the diver gradually equilibriates where the same amount of D2O and H2O is transferred. On log graph there is a gradual plateau. 
Rapid exchange of cell water in all cases with the t½ of exchange < 4.5 minutes bar In trout eggs no evidence of water diffusion even after 5 hours
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How to measure Pf?

A

Meaure the change in volume of a cell overtime when exposed to hypertonic/hypotonic solutions.

Pf= AV/ SA x t x AC
Change in vol (diameter)= Pf x Surface area x time x concentration change (gradient)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

1953 cell study Pf?

A

All cells swelled when added hypotonic solution in the bath (low solutes outside- water rushes in the cell) and shrinks when hypertonic (increased solutes outside- water leaves).
All cells bar the trout egg.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Explaination for trout eggs very low (no) water permeability ability?

A

They are layed into freshwater (not saltwater), so likely evolved to lose this ability else water will just rush in and the egg will swell and burst.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Red blood cell water permeability is very..? Therefore..

A

High.
Pf/Pd ration =2.5.
This predicts a water pore in. (was very hard to isolate)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How was the RBC water pore eventually found? (part 1- until AB to)

A

Agre group found 1991:
Was studying the Rheus protein in RBCs but kept getting a 28kD proein co-precipitating. (1988)
Isolating this protein was hard but producted an antibody to the 28kD protein.
This AB also recognised a higher MW band (but not the 32KD rheus protein) and 2/3 times the size (exists as tetromers- cryoEM confirmed).
If treated with a deglycosylation enzyme- drop back to 28kD.
AB stained PT and thin decending limb (high water permeability)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How was the RBC water pore eventually found? (part 2- until calcualtions)

A

Agre group 1991:
The N terminus of the 28kD protein was sequenced (from human faetal liver) so PCR and library screening could be used to identify CHIP28 (AQP1).
Sequence analysis predicted 6TMD and 42% homology to waterchannel in the eye (AQP0- makes up 60% of lens is).
Both had tandem NPA repeats (suggested critical form
water pore formation).
Biophysical Calculations of water channels needed and number of CHIP28 in RBCs added up. And both resistant to enymatic digestion.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How was CHIP28 (AQP1) proven to be a water chanel?

A

1992:
CHIP-28 was expressed in xenopus oocytes and then the oocytes exposed to a hypertonic shock
In control oocytes the volume change is very slow.
In oocytes expressing CHIP-28 the volume change is rapid and the oocytes explode with in a few minutes. Renamed AQP1.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

AQP1 is inhibited by? Ecidence?

A

Mercury sensitive.
1993 study.
Measured the volume of xenopus oocytes expressing AQP1 when exposed to hypertonic shock (water inflow) . Volume increase of 35%.
If add Hg- volume increase decreased to 15%.
If add ME (beta Mercatoethanol reducing agent which binds to cysteins so mercury cant) back to 35%.
Xenopus without AQP1 expression vol increase of 5%.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How does mercury inhbit AQP1? Evidence (2)? (not specific number)

A

Mercury binds to exposed cysteine residues of AQP1.
Know because treatment of B Mercatoethanol binds to cysteins so HgCl2 cant stopping the Mg inhibition (volume returns to 35% increase after 15% with Mg).

Also, If mutate 4 cysteins in the AQP1 and replace any with serine the water permeability increases after Hg treatment compared to when cysteins (cant bind and inhibit) But no impact on water permeability without Hg.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

WHich aquaporin is mercury insensitive?

A

AQP4.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Exposed cystein that Mercury binds to? Evidence? (x2)

A

Cystein 189.
1993 Preston: If mutated cystein residues individually and see impact on mercury on the water permeability.
Results:
Normal function of AQP1 without HgCl2.
HgCl2 still reduced AQP1 function with mutated 3 of the cysteins, but not C189.

Savage 2007 study: Also.. If take bacterial AQ1 (HgCl2 insensitive) and engineer a C189 to it- forms crystals with Hg and now mercury sensitive.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

AQP1 stucture?

A

6TMDs, with the B and E (between 2-3 and 5-6) loops not crossing the membrane but dipping into. These have the NPA motifs.
These two dipping loops cross over each other and make the pore between. This creates an hourglass like model.
4 subunits come together to give the tetromer- each making a pore through.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

AQP1 have 4 pores or one central? Experiment method?

A

Made of 4 subunits coming together (6TMDs and have a pore in each).
Jung 1994. Made tandem dimers of either AQP1 subunits or C189s mutants.
mRNA injected therefore will be mix, could be all WT, all mutant or mix etc.
If mix would it give an intermediate phenotype ie 3/4 mutant- have 3/4 pores working etc, or if central pore will be all or nothing function.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

AQP1 have 4 pores or one central? Experiment results?

A

Yung 1994-
Without mercury all channels gave functional water channels.
WT- mercury brings back to baseline levels.
Gave intermediate (25%, 50%, 75%) mercury sensitivities when mixed WT and mutant C189S subunits.
Concluded: Must be individual functional channels

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

WHat is surprising about how water moves through aquaporins?

A

There is no piggybacking of protons through, which would be expected, however there is a single file movement of water through the pore.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

How is water transported in single file through aquaporins? Model for?

A

Predicted that watermolecules H+ bonds are broken and they bind to the NPA motifs through new H+ bonds which ensures selective water movement through which inhibits protons piggybacking.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Experiment of single file water movement through AQP1? Method:

A

NSF motifs in B (Asn-Pro–Ala) and E and a hydrophobic lining of the pathway for the water through.
Stochastic boundary molecular-dynamics (SBMD) method was used to probe the dynamics of particles ad the H2O moved through.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Experiment of single file water movement through AQP1? Results:

A

It was seen that the distribution of water density near the NPA motifs is significantly thinner, confirming that the narrower space around that region imposes a constriction to the flow of water.
Also hydrogen bonds with the polar (hydrophillic) side chains of NPA could be seen on the stereochemistry diagrams and were found to be essential. When in B loop the Asn and Asn were replaced with near-identical hydrophobic residues in the simulation, the aqueous pathways were broken completely.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Polar is hydrophobic or phillic?

A

Hydrophillic. (polar bears love swimming in water to get fish)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Experiment of single file water movement through AQP1? Discussion:

A 
Discussion: 
The size restriction on molecules to pass by the narrowing through the NPA will limit what goes through the channel and promote the single file pasage.
Importantly, the size of the constriction fluctuates significantly, which frequently breaks the flow of water, thus breaking the single-file water network necessary for proton translocation
The polar(hydrophillic) NFA residues are essential to H bind to the water molecules and ensure H+ ions cannot pass alone.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

AQP3 channel for? Found?

A

If express in xenopus oocytes, increase the mercury sensitive passage of water through.
Also a Hg++ sensitive increase in glycerol and urea permeability. Could measure when radiolabelled.

Found: In basolateral collecting duct to reabsorb water for example.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

AQP6 channel for? Found?

A

Unorthodox aquaporin.
Found in the kidney and colocalises with H+ ATPase proton pump in in alpha intercalated cells.
MERCURY STIMULATED.
water permeability increases with. Cl- conductance also.
PH SENSITIVE also- stimulated at low PH ( if H+ in (acidic) Cl- will also come in to balance charge)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

AQP6 stimulated by?

A 
Mercury. 
Low PH (acidic- Cl- in to balance charge)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

Classes of MIP proteins?

A

1) Aquaporins e.g. AQP 0,1,2,4,5 (water only)
2) Aquaglyceroporins e.g. 3, 7,9, 10 (+glycerol and urea)
3) Unorthodox e.g. 6, 8, 11, 12 (NPA changes to NPS)
13 in humans.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

AQP8 channel for?

A

linked to ammonia transport

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

AQP2 mutation?

A

Diabetes insipidis.
Cannot concentrate their urine- lose 20l urine a day.
Vasopressin doesn’t help (inserts more AQP2 into the apical membrane of the collecting duct.
These mutations may affect trafficking or permeability to H20.
AQP3 and 4 on basolateral.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Aquaporins in epithelia?

A

Reabsorptive Epithelia – Proximal tubule – AQP1

Secretive Epithelia – Salivary Gland – AQP5

Reabsorptive Epithelia – Small Intestine – No AQPs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Importance of AQP1? Study

A

Hand dissected out kindey and mount on micropipettes. and measure water permeability.
KO AQP1 in proximal tubule: water permeability decreased by 78%. (Pf)

water cant move across to balance the hypotonic gradient in lumen created, therefore the gradient is much larger (from around 10mOsmol/L to around 40mOsmol/L)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

How does water reabsorption in proximal tubule occur?

A

At near iso-osmotic conditions.
Na and Cl out of the lumen into the epithelial cells, leaving the lume slightly hypotonic. Water follows and the epithelial cells reabsorb water in through AQP1 (basolateral). But extremlely high water permeability of the tubule means this is sufficient.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

PCT cell what channels are on Apical and basolateral?

A

Apical: Na/H exchanger and H recycled back in. CO2 diffuses in.
Basolateral: Na/HCO3 cotransporter and AQP1.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

How does microperfusion experiments occur in PCT?

A

Dissect out PCT with the outer of epithelial cells and inner lumen. Liquid in lumen= perfusate (what is added) and liquid outside the epithelial cells=absorbate (what is reabsorbed from PCT).
Oil droplet is put around the vessel on basolateral side of ECs and this makes up the absorbate. As the cells reabsorb, a water droplet forms under the oil droplet= absorbate.
Can remove this oil and measure its osmolality difference between the two. Meausre H2O gradient.
around

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

Experiments to prove PCT water reabsorption near-isotonic?

A

16mosmol/kg (near iso-osmotic) PCT H2O gradient difference from perfusate to absorbate using microperfusion.

Micropuncture experiment-

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

Micropuncture technique?

A

In vivo. Pipettes put into capillaries of anaethasised animals. Known NaCL (no HCO which also drives water reabsorption though) solution concentration into PCT lumen and capillaries and the PCT fluid was collected.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

micropuncture technique results?

A

Less than 2mM gradient drives lots of water reabsorption.
Low flow rate: slightly hypotonic PCT fluid collected compared to the capillary.
High flow rate: rate of absorption higher.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

Why is transport in the PCT slower in high flow rates?

A

Fluid comes to equilibrium in the PCT, at slower flow rate reaches equilibrium faster (gradient neutralised), so no more fluid can be absorbed (e.g. Gordon cooper walkign across lecture us with paintguns, shoot him all over if going slowly, less if fast.)
Whereas in slower flow rates, gradient persists for longer and so more water reabsorbtion can occur.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

Secretive epithelia example?

A

Acinar cell.
Salivary gland.
Has AQP5.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

secretive epithelia how works?

A

Secretes Na and Cl- into lumen- creating a hypertonic lumen. This creates an osmotic gradient for water out of the cell through AQP5 (balance out).
Uptake on basolateral through NKCC1 (Na and Cl- into cell) and Na paracellularly.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

AQP5 KO?

A

Measured salivary secretion after Pilocarpine (stimulates salivary secretion) Measure vol of secretion and osmotic conditions.
WT- high secretion, osmalarity near isotonic. (water balances)

AQP5 KO- less than half and high osmolality- highly concentrated saliva (low water content)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

Whats odd about water reabsorption in the small intestine?

A

Apical side lacks any aquaporins and is quite tight. Has a high osmolality (500-550 mOsm) in lumen as releasing Na, Cl, AA, proteins after food broken down. This would drive water secretion. But uphill reabsorbtion against gradient (200mOsm gradient). SOMETHING MUST BE ACTIVE.
Some transporters also transport water.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

Hyper and hypotonic?

A

Hypertonic- high solute concentration, high osmolality- water will move into.
Hypotonic- low solute concentration, low osmolality, water will move out of.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

Epithelia that are reabsorbing water? Secreting?

A

Reabsorbing: Kidney PCT CD, small intestine
Secretory: airway

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

Cotransporters that also transport water?

A

KCC4- 500molecules of H2O every pass
NKCC1- 590 (basolateral of cells e.g. salivary)
GLUT2- 40-100

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

NKCC1 or 2?

A

1- basolateral of cells e.g. salivary glands (has water transport role)
2- kidney e.g. thick ascending limb

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
50
Q

Study evidence for water transport with other cotransporters?

A

Frog cell expressing KCC transporter, measure cell shrinkage/growth after hypotonic shock (graidnt increase 100mOsm) with mannitol 100mM).
generally cells shrink as water moves out.
Add NaClmM (100mOsmol change) shrink again.
Add 50mM of KCl. Now cells swell. Because KCl wants to move into the cell against gradient and water comes in with the KCC4 channel even against the osmolality gradient.
And Furosamide (Inhibit KCC and NKCC proteins), cell shrinks again, so is through this channel.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
51
Q

Water moves throuh aquaporins through.? Through other channels by?

A

Aquaporins: Diffusion.
Channels: cotransportation
Mix of both, SGLT EAAT1 (amino acid transporter)

52
Q

Which channels have a mix of diffusion and cotransportation through channels? How?

A

SGLT (Na glucose cotransporter)
EAAT1- amino acid transporter
Creates microgradients where transport solutes across in very thin layers next to transporters, creating gradients of water. So say sodium moves into cleft, creates gradient and water will move in wth glucose- conformational change and moved across.

53
Q

Water reabsorption in the small intestine channels?

A

APICAL: 500mOsm. SGLT2 brings in Na, glucose, H2O and GLUT2 brings in Glucose and H2O from the lumen.

Basolateral: 300mOsm. Na/K ATPase(Na out, K in). KCC (K recycles, Cl- into the blood as well as H2O), GLUT2 also brings glucose and H20 in reabsorbing.

54
Q

AQUAPORIN1 also has a channel for..? relevence?

A

Gas.

Red blood cells and colton null people important in

55
Q

What was thought before Overtons law? Why?

A

It was previously thought that membranes were fully permeable to solutes/gases.
Because O2 or CO2 have a high solubility in oil so are freely permeable so it was presumed that all membranes were freely permeable to all gases.
Also, artificial membranes were freely permeable but these had no cholesterol or membrane proteins and decane preparation solvent increased fluidity.

56
Q

What is overtons law?

A

Overton realised the permeability of membranes to gases/solutes was proportional to the oil/water partition co-efficient for that solute.

57
Q

Experiment to test overtons law?

A

See how well different solutes dissolved in oil vs water to test the theory that permeability of membranes to gases/solutes was proportional to the oil/water partition co-efficient for that solute.

58
Q

NH3 causes cells PH to?

NH4 causes cells PH to?

A

NH3(ammonia)- PH rises (alkaline) as NH3 combines with free H+ ions in the cell to become NH4.

NH4(ammonium)-PH falls (acidification) as NH4 dissociates into NH3 + H+. But slower uptake of ammonium into cells, as isnt permeable to.

59
Q

Are membranes less permeable to ammonia or ammonium?

A

Ammonium (NH4), Isnt permeable to across bilayers, but is transported through transporters like NKCC (in thick ascending limb) instead of K.

60
Q

CO2 or HCO3 into a cell impacts PH how?

A

Co2 moves into cells, combines with H2O= HCO3+H+ (acidification)
HCO3 moves into cells, combines with H+ to make CO2 and H20- uses up free H+, alkalisation.

61
Q

If cell there is acidification transported in is? (x2)

Alkalisation? (x2)

A

Acidification: Ammonium NH4 or CO2.- (generates H+)
Alkalisation: Ammonia (NH3) or HCO3

62
Q

If cell there is acidification transported in is? (x2)

Alkalisation? (x2)

A

Acidification: Ammonium NH4 or CO2 (generates H+)
Alkalisation: Ammonia (NH3 gas) or HCO3-

63
Q

First experiment that evidenced low gas permeability?

A

Permeability to NH4/NH3 in thick ascending limb.
Measured the PH of cells, and if Ammonium chloride is added to the basolateral side see an alkalisation, predictable permeability to ammonia and ammonium.

but to the apical see an acidification, high permeability to NH4 but very low NH3 (gas).

64
Q

Follow up experiment that evidenced low gas permeability?

A

Gastric gland permeability to CO2/HCo3.
Dissected out gastric glands and put on micropipettes
if Co2 put on basolateral side- acidification (as expected)
but if 100% Co2 added on lumen side- no PH change- impermeable to gas.
Same result in colon crypt.

65
Q

Why may the gas impermiability make sense in gastric glands?

A

Stomach PH very low HCL- near to 1 very acidic.
So having the apical membrane impermeable to gases is protecting stomach from digesting itself, else the cells would become hugely acidic and not be able to survive.

66
Q

What causes low gas permeabiltiy of membranes? Experiments (2)?

A

Linked to cholesterol content.

  1. Experiments using liposomes artificial bilayer, can change the cholesterol content in.
    - At low cholesterol levels: High Co2 permeability freely
    - As increased into physiological range- linear decrease in Co2 permeability.
  2. MDCK kidney cultured cells, measured CO2 permeability. If strip out cholesterol from membrane, CO2 permeability increased massibvely, and if increased- reduced.
67
Q

Cholesterol content level in thick ascending limb? Collecting duct?

A

TAL: predict v high cholesterol due to low gas permeability.
CD: high cholesterol but aquaporins so tight but regulated passage.

68
Q

Cancer cells cholesterol content?

A

Low (30%), as they produce Co2 which needs to be lost and O2 to diffuse in to meet metabolic demands (very active cells).

69
Q

Colon crypt cholesterol content?

A

77% cholesterol in apical as well as proteins in there to reduce surface area for gas movement. Glycolipids and glycoproteins to reduce also.

70
Q

RBCs cholesterol content?

A

Apical: 45% but this wouldnt support the amount of Co2 transport that has been measured. Proteins and transporters also.

71
Q

Proximal tubule cholesterol content?

A

Apical: 45% but this wouldnt support the amount of Co2 transport that has been measured. Proteins and transporters also.

72
Q

WHy use the xenpus oocytes in experiments into Co2 permeability?

A

If add a Co2/HCO solution know its gas movement as there are no native bicarbonate transporters in the xenopus oocyte. = Gives acidification and this is proportional to the CO2 permeability (without bacground bicarbonate permeabilityy).

73
Q

How to measure CO2 permeability in Xenopus oocytes?

A

If add CO2/HCO solution (acidification) and then add water, can measure how long it takes the cell to swell and explode.
Co2 permeability is proportional to water permeability. The more AQP1 channels there were (and faster lysis time), the faster the acidification was.

74
Q

what factor can you use to measure CO2 permeability in the xenopus oocyte (not water)

A

If add a Co2/HCO solution know its gas movement as there are no native bicarbonate transporters in the xenopus oocyte. = Gives acidification and this is proportional to the CO2 permeability (without bacground bicarbonate permeabilityy).

75
Q

How can one check the Co2 permeability experiments in xenopus oocytes are not just the aquaporins making the membrane more leaky?

A

If add an organic mercurial agent which inhibits the AQP1, CO2 permeability decreases. Also repeat this with C189s mutant, the CO2 permeability is returned with mercurial agent.
First experiment proving that gas permeability is linked to protein channels.

76
Q

what are the group of people called with no AQP1?

A

Colton null blood group

77
Q

How much of CO2 permeability is due to AQP1? How do we know?

A

Colton null blood group donated some RBCs.
These lack AQP1. CO2 permeability was measured and this was reduced by 50%.
So roughly 50% of the CO2 permeabilty is due to AQP1. ( using mercurial agent had no impact also)

78
Q

As well as AQP1 how else is CO2 passing through RBC membrane? Experiment?

A

DIDs blocks CO2 permeability through AQP1 but not water permeability.
In WT- reduced this by around 1/3.
Colton null- reduced the Co2 permeabiltiy even further than already was (50% of WT already).
DIDs inhibits rheus protein in RBCs- so around 40-50% due to this.
10% left through lipid bilayer

79
Q

What goes through each pore in AQP1?

A 
x4 outerpores (in each subunit) Water and Co2(40%). Have both hydrophillic and hydrophobic domains in. 
x1 central pore- Co2 (60% ) and O2 (85% through central , 15% through outer). Mainly hydrophobic and gated by hydrophobic residues which prevent water access.
80
Q

AQP4 permeability to Co2?

A

M1- Kidney- has no permeability (in full length protein whereas in truncated M23 does)
M23 in BBB- Co2 permeability- this will help with gas movement as this an extemely tight.

81
Q

Under what chemical can AQP1 not become CO2 permeable.

A

AQP1 binding of cGMP to, turns it into an anion channel throught the central pore.
Oocytes treated with cGMP, AQP1 or 5 will lose Co2 permeability. cGMP changes conformation so CO2 cant go through but cations can- but no impact on water permeability. REGULATION.

82
Q

AQP5 where? function?

A

Also has Co2 movement through. Expressed in type 1 pneumocytes in the lungs to allow gaseous exhange into/out of the blood.

83
Q

Small intestine has no water porins yet it reabsorbs… liters of fluid daily. How?

A

Around 8 liters a day.

SGLT1 can cotransport water with the ions.

84
Q

Method to measure water through SGLT1?

A

We overexpressed SGLT1 (eGFP tagged) in MDCK cell monolayers and reconstituted the purified transporter into proteoliposomes. We observed the rate of osmotic proteoliposome deflation by light scattering i.e. if they deflate water has left the liposome. After measuring the SGLT1 abundance on MDCK cells and concentrations either side of the monolaters membranes, the unitary water channel permeability, pf was measured. This yielded similar values for cell and proteoliposome experiments so was deemed a suitable model.

85
Q

Aim of the SGLT1 water through experiment?

A

Previously suggested that high solute amounts passing through the SGLT1 channel would inhibit water through. But also wanted to test whether no water could still pass without Na or glucose. AIm to find out how they are all transported in the channel, together or separate?

86
Q

Result of the SGLT1 water through experiment?

A

Neither the absence of glucose or Na+, nor the lack of membrane voltage in vesicles, nor the directionality of water flow grossly altered pf. Not the limiting step for water movement through?
Addition of Na had no impact on the Pf but 20 mm glucose addition resulted in a 17% drop in pf , suggesting that at least some of the water molecules share the pathway of glucose and are delayed by its presence.

87
Q

conclusion of the SGLT1 water through experiment

A

Such weak dependence on protein conformation indicates that a water-impermeable occluded state (glucose and Na+ in their binding pockets) lasts for only a minor fraction of the transport cycle or, alternatively, that occlusion of the substrate does not render the transporter water-impermeable as was previously suggested.
Water partially shares the glucose route, and thechannel is permeable to water when empty, and with solutes (definitely Na). The high Pf levels of the SGLT1 excludes the need for active pumping in the small intestine inspite of against the gradient.

88
Q

Proximal tubule PH? Why?

A

PCT PH 7.4.

with Plasma HCo3 around 25mM and 5% Co2 (1.2mM dissolved).

89
Q

Most of the reabsorbed HCO3 from kidney is where?

A

80% in the PCT.

90
Q

APical movement of HCO3 PCT reabsorbtion is due to?

A

CO2 reabsorption across the PCT and H20, which cab combine to make HCO3 inside the cell.

91
Q

How is HCO3 reabsorbed over the PCT?

A

In lumen HCO3- (+H) to make carbonic acid, which then dissociates into H2O and CO2 under carbonic anhydrase. Co2 can move across the membrane (CO2 in AQP1). water already in.
When inside the cell these combine to make HCO3- + H+ (carbonic anhydrase). The H+ recycles across the membrane by NHE3 exchanger (out) and active proton pump into cell.
THe HCO3- then leaves over the basolateral side with Na (1Na: 3HCO3-)

92
Q

Where are the different carbonic anhydrases?

A

Type IV in lumen of PCT. Tethered to apical membrane.

Type II in PCT cells.

93
Q

NHE3 stoichiometry important why?

A

1Na: 3HCO3- NHE3 in PCT- need for reabsorption.

Whereas in pancreas 1Na: 2HCO- drives uptake of 2HCO3- into the cell

94
Q

What factors stimulate HCO3- reabsorbtion?

A

Angiotensin II
Endothelin 2
NorA, Adenosin
ALL INCREASE CA AND CAUSE PKC ACTIVATION

95
Q

What factors downregulate HCO3- reabsorption?

A

ANP
Dopamine
Perathormone
INCREASE CAMP- PKA ACTIVATION

96
Q

If high levels of … in blood, HCO3- reabsorption is..

A

CO2 high in blood, HCO3- reabsorption is inhibited

The level of CO2 in blood regulates HCO3- reabsorbtion across the PCT.

97
Q

Experiment to test whether CO2 is regulating HCO3 reabsorption across the PCT?

A

Out of equilibrium Co2/HCO3- solutions. Add pure Co2 and no HCO3- or pure HCO3- and no CO2.
If add HCO3- going to get Co2 as dissociates etc, so wanted to design a solution which were at 100% at 7.4PH (out of equilibirum where normal PH 7.4 blood 5% CO2 and 22mM HCO3-)

98
Q

How were the out of equilibrium solutions made?

A

Solution 1:
a. Solution at 10% Co2 at acidic PH (5.4), V low HCO
b. Other syringe 100% oxygen, no Co2 or HCO- PH 7.55 but heavily buffered.
MIX with mesh (turbulent flow)= Pure Co2 PH 7.4 normal 5% Co2 and no HCO for few hundred milisecond.

Solution 2:
a.  slightly acidic solution of no CO2 or HCO.
b. 44mM HCO3 of alkaline PH 
MIX= Pure HCO3- 7.4 PH. 
(NaOH buffer ensures no CO2 dissolved)
99
Q

Pure CO2/HCO out of equilibirum results?

A

Used the pure CO2 or pure HCO3- solutions to run through PCTs to compare reabsorbtion solution on basolateral membrane.
Removing HCO3- increased HCO3 reabsorption (when normal Co2). Increase driving force and preventing backflux through tight junctions.

When no Co2- HCO3 reabsorption decreased. Basolateral blood CO2 sensor in PCT?

100
Q

CO2 sensor for HCO3- reabsorption works how?

A

Basolateral PCT Co2 sensor. If chnage concentrations in lumen- no impact but change in bath Ca increased in PCT cells.
Co2 sensor on basolateral side (in blood).
Repeated with pure HCO3- no change in Ca, but added pure Co2- got change in Ca, so looks like this is what is being sensed.

101
Q

Possible identity for the basolateral CO2 sensor in PCT?

A

Receptor protein tyrosine phosphatase Y (membrane linked).

102
Q

Evidence that it is Receptor protein tyrosine phosphatase Y that is the PCT basolateral CO2 sensor?

A
  1. If this is inhibited by Receptor tyrosine kinase inhibits, the link between CO2 levels and HCO3- reabsorption was abolished.
  2. Localisation experiments-present in the PCT at basolateral membrane.
  3. -/- KO RPTP mouse- look at HCO3- PCT transport. Fixed PH and HCO3- but changed CO2 levels- inhibited HCO3- transport generally. And this linear relationship was lost. HCO3- changes no reponse either (WT does)
  4. Phosphatase binding domain faces towards blood has homology to carbonic anhydrase site where CO2/HCO3- can bind to.
103
Q

Physiological reason for HCO3- reabsorption control by CO2 in blood?

A

High Co2 in blood- in acidic condition(acidosis). If reabsorb more HCO3- across PCT- balance Ph (HCO3 can sequest H+)

104
Q

Theory of how the CO2 HCO3- PCT sensor works?

A

CO2 and HCO3- binding to receptor protein tyrosine phosphatase Y- inhibition of its phosphatase activity- the Receptor protein kinases activity will dominate.

105
Q

Why is bicarbonate transport important?

A

Helps drive water reabsorption.

Buffers PH.

106
Q

Bicarbonate cycle of dissociation etc?

A

HCO3- + H+ = H2CO3 (carbonic acid).

H2CO3= CO2 +H2O dissociates under carbonic adhydrase enzyme.

107
Q

Method for measuring link between HCO2- reabsorption and water?

A

Rabbit PCT, injected an oil drop into the PCT lumen (stop perfusion either end of tube), and inject fluid in the middle (can change composition). Measure volume of this fliud drop in PCT, will shrink as cell reabsorbs water etc. (split drop micropuncture technique). Proportional to water flux Pf.

108
Q

Results for measuring link between HCO2- reabsorption and water?

A

If high HCO3- composition in lumen- high water reabsorption, whereas if remove the HCO3 the shrinkage of the waterdroplet in the lumen decreased.

109
Q

Role of the NHE3 in HCO3- reabsorption?

A

KO mouse for NHE3 (Na and HCO3- over basolateral membrane to reabsorb in PCT).
Microperfusion technique saw a decrease in plasma PH (more acidic as HCO3- decreases so doesnt sequest H+ ions). HCO3 transport down by 60%. Water flux also decreased by around 70%.

110
Q

NHE3 in PCT drives ….. HCO3- reabsorption and ….. water.

A

60%

70%

111
Q

HCO3- is ….in the pancreatic duct?

A

secreted

112
Q

Model of pancreatic duct for HCO3- secretion in rats?

A

On apical membrane- Cl- HCO3- exchanger secreting HCO3-. Cl- is recycled as it is pumped back out through CFTR.
On the basolateral membrane- NHE (Na in). Co2 moves through, combines with H2O- HCO3- leaves across apical and H recycled across basolateral.

113
Q

Why is the pancreatic secretion model different for other mammals that arent rats?

A

MOre simple in rats as their lumenal HCO3- is only 70mM, but other mammals upto 140mM (humans ginea pigs etc)- electrochemical gradient to move back into cell, Cl/HCO3- shuts down.

114
Q

How is the HCO-3 secretion in non rat mammals achieved?

A

Against electrochemical gradient, Electrogenic Na/HCO3- cotransporter- Na 1: 2HCO3- into the cell. Brings into the cell over basolateral membrane, building concenration up in cell so favour secretion. Also HCO3- secreted out through CFTR.

115
Q

Evidence for apical secretion of HCO3- through CFTR in pancreatic duct? 2009 Method:

A

Isolated the pancreatic duct in WT and AF508 mice. Controlled luminal and baso contents and measured the PH inside cell.
If HCO3- moves into cell= Alkalisation (binds to protons)
If HCO3- moves out of cell= acidification.
Set up solutions: Baso Cl- and HCO free and added DIDS (inhibit Na/HCO3-) and CAMP to stimulate CFTR.
Lumen CL- free but high HCO3- concentration.
Step changed K concentration (changes membrane potential)

116
Q

Evidence for apical secretion of HCO3- through CFTR in pancreatic duct? 2009 Results:

A

Increased K conc- depolarised the cell, HCO3- moved through CFTR (secretion) = Acidification.
If hyperpolarise-HCO3 moving in= Alkalisation.
Repeat in AF508- no PH changes at all-therefore Ph changes driven by HCO3- movement through CFTR.

117
Q

As go down the pancreatic duct how does secretion were?

A

Acinar cells secrete a Cl rich secretion. NKCC1 in basolateral causes Cl uptake, and Cl and Na secreted across the apical membrane (sectreted) As fluid moves down the duct Cl is reabsorbed by intercalating duct cells and replaced by HCO3- secretion.

Early in duct SLC26 (anion exchanger including Cl- in, HCO3- out) and CFTR (HCO3 and Cl out) combination on apical membrane.
But CFTR inhibits CL/HCO30 exchanger further down the duct and HCO3- is mainly secreted.

118
Q

CFTR and SLC26 interactions?

A

SLC26 interactions with CFTR.
At rest- both inactive. CFTR NT binding domains inhibiting CFTR.

With PKA stimulation, P of regulatory domain, CFTR inhibition is released so activated.
Interaction with regulatory domain of SLC26, which promotes the Cl/HCO3- exchange (recycling of Cl- acorss membrane while secreting HCO3-).

119
Q

Why is SLC26 not simply working to secrete HCO3- in experiments?

A

EXperiments were done in CL- free conditions, and this needs Cl- to exchange- so its CFTR mainly.
Also CF mutations affect HCO3- transport.

120
Q

Further evidence of CFTRs role in transporting HCO3- and that it is a different mechanism to Cl-?

A

CF patients with pancreatic insufficiency or sufficiency were studied.
If stimulate WT CFTR with forskolin get Cl- movement= current. Also, PH change as HCO3- secreted.

I148T mutation- normal Cl- transport but HCO3- disrupted- insufficient.
R117H- slight Cl disruption but HCO3- retained= sufficient.

121
Q

NBC?

A

The electrogenic sodium bicarbonate cotransporter

122
Q

NBC splice variants?

A

kNBC: Kidney PCT- 1Na into blood: 3 HCO3 into blood -ABSORBTION HCO3-
pNBC: Pancreatic- 1Na into cell: 2 HCO3 into lumen- SECRETION HCO3
(N terminus difference)

123
Q

Does the N terminus account for the stoichiometry difference of NBC? Method:

A

Compared a PCT cell line (reabsorb) and cortical collecting duct cell lines (secrete) with no NBC activity. And expressed pNBC and kNBC in both transfection. Mounted in using chambers and voltage clamped.(electogenic IV relationship). The reversal potential was measured when the Na gradient was varied.

124
Q

Does the N terminus account for the stoichiometry difference of NBC? Results:

A

PCT cells: Vrev= 20mv (when Nai=0 and Nao=50)
Stoichiometry =1Na: 3HCO.

CD cells: Vrev= 40mv = 1Na: 2 HCO.

Therefore, its not the N terminus that determines the change in stoichiometry, but where the channels are expressed.

125
Q

What does cause the stoichiometry difference in the NBC?

A

Tested Protein kinase phosphorylation. Before 1:3, after 1:2.
In a mutant with an inactive PKA site, the channel was stuck at the 1:3 stoichiometry.
In a mutant that mimics phosphorylation, stuck in the 1:2 stoichiometry.
PKA stimulates HCO3- secretion.

126
Q

NBC stoichiometry changes with phosphorylation, but also what? Know how?

A

Calcium levels.
In xenopus experiments:
- low Ca- Vrev -70, 1:2.
-High Ca- Vrev -40, 1:3.

Calcium stimulates HCO-3 reabsorption.

BMS 109 (20 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions