GC lectures Flashcards
Diffusional water permeability (Pa)
permeability to water when there is no osmotic gradient
rate= diffusional water permeability
influx=efflux
What does water balance under?
Water basic isotonic conditions- no net movement
Hypertonic / hypotonic solutions
hypertonic- cell shrinks
hypotonic- water moves into cells and it swells and bursts
Pf/Pd ratio
if this ratio is 1= then as water pore is present in the cell - water moves along the gradient
Which model do they use to measure the Pf/Pd ratio
Measured using a variety of cells, xenopus egg, amoeda, trout egg, zebra egg, frog egg
Why are they the model choices?
All spherical, easy to measure + diameter
large and easy to work with
Measuring Pd- cartesian diver balance
- diver rises- air bubble gets bigger
- air bubble gets smaller- diver sinks
* measure pressure needed to keep the diver @ a constant height
Technique used to measure cartesian diver balance
Small glass funnel- place cells in it
Top= air bubble
Balance air bubble and height- stays in water
Apply air pressure- air bubble gets smaller- less boyant- sinks down into solution
diver balance in D2O
Cells equilibriated in H20
cells placed in solution containing D20 (heavy water)- makes diver heavy
D20 exchanges with H20 in cells
cells become heavier- diver starts to sink- apply suction to keep the diver at a constant height
What is change of EP correlated to
Change in weight- from this change in cell weight can be measured then it is known how much D2O has moved into the cell and the Pd is calculated
Pd conclusion
Rapid exchange of cell water in all cells with the t1/2 of exchange <4.5 minutes for the cell types except for trout egg cells (5 hours)
*no evidence of D20 entering the trout cells
Measuring Pf
measure change in cell volume over time when exposed to a hypertonic or hypotonic solution
Equation to measure Pf using hypertonic or hypotonic solution
change in vol= Pf x SA x T x change C
Results of measuring Pf
All the cell types (besides trout) were a Pf/Pd ratio >1
indicates that these cells have some kind of water pore in their membrane except the trout cells which have no water permeability
osmotic water permeability drops if its put in water
Zebrafish in low solution
Cells explode
All laid into fresh water- osmotic permeability plummeted
protective mechanism
Red blood cells permeability
Osmotic permeability= 1.5 x 10-14
Diffusional permeability= 5.3 x 10-3
Equivalent to 0.64 x 10-14
Pf/Pd ratio for RBCs
2.5- water pores/ some kind of water pore
prediction in this study that it would be 3.5A
How many aqua1 are there in RBC
200,000
Aqua1- functional characteristics
CHIP28
mercurial sensitivity of aqua 1
structure and functional unit aqua1
aqua1
Identified by group of peter andre at john Hopkins
initially studies involved rhesus proteins
kept finding 28kd proteins that coprecipitated with a 32 Kd Rh polypeptide
isolated the 28kd protein and produced an antibody
What did the antibody show
recognises a 28KD Protein, and a higher mw band, never found any labelling of a 32kd protein
the 28kd and 32kd proteins are not related- doesn’t show up in traditional statins
What is the 32kd HMW band appearing to be?
High glycosylated form of the 28Kd
evidence the protein exists as oligomers in the membrane
Antibody staining in the proximal and distal thin limb
Band= probing with antibody- 3x4 times the size of the subunit
High MW version= high glysolyated
treat with PNGA- drops to anginal protein level
Cloning of aqua1
N terminal portion of the 28kd protein was sequence
with this information a combination of PCR and library screening was used to identify the message for CHIP28
What did sequence analysis predict
A protein with MW 28kd
What do we know about the structure of the water pore for sequence analysis?
6TM spanning domains
42% homology to MIP26 and high homology to several proteins with no known function
all related clones have tandem repeat of aa sequence NPA
speculate CHIP28 involved
Is CHIP28 as water channel- circumstantial evidence
actual copies of CHIP28 IN RBC and biophysical calculations of channel number are in the same region
28.5kd unit similar to 30kd functional unit of proximal tubule water channel
CHIP 28 transcript corresponds to RNA fraction of Kidney that produces greatest water channel activity
What is CHIP28 resistant to
Ezymatic digestion as in the RBC
experimental ecidence that CHIP28 is aqua1
CHIP28 was expressed in xenopus oocyte and exposed to hypotonic solution
in control oocytes the vol change is very slow
in oocytes expressing the CHIP 28 the vol change is rapid and oocytes explode in a few mins
What does CHIP28 do?
Confers high water permeability on RBCs and the proximal tubules
renamed aqua1
Aqua1- mercurial sensitivity? why do we think this
RBC is very sensitive to HGCl2 or the organic mercurial PCMBs
Experimental approach to see if aqua1 is mercurial sensitive
pre incubation in HgCl2 has no effect upon control oocytes
pre incubation in HgCl2 slows the volume change in oocytes expressing AQP1
The reducing agent B meracathathanol reversed the Hg induced inhibition
Result of the mercurial sensitive experiment
Express CHIP28- rapid change in volume when exposed to a hypertonic solution
When you add mercury H20 is reduced
= mercury sensitive
What is the basis of mercurial action
Mercurial agents exert an action by binding to cysteine residues
AQP1 contains 4 cysteine residues at amino positions 87,102,152 and 189
mutate each of the cysteine resides individually to serine- test effect on HgCl2 water permeability to these mutants
Results from mercurial action- mutating cysteine residues
Mutants have the same water permeability as WT aqp1
HgCl reduced water permeability in C87s, C102s and C1525S but had no effect on C189 s mutant
*conclude Hg binds 189
What do the mutants affect
Don’t disrupt function of water pore
all water pores + osmotic permeability don’t change in wt but change interaction with mercury
hourglass model
Amino and carboxyl ends are IC
6tm domains
loops between 2-3 and 5-6
as protein folds NPA motif come to lie together producing channel- hourglass
What shape is aquaporin 1 and how is it confirmed?
tetramer
early studies indicated that aqp1 formed as a tetramer in the membrane
confirmed by cyro-electron microscopy work
Small region indicates where NPA motifs overlapping, looks like pore in each region and has central pore
Discovering if aqp1 is a monomer or tetramer? Approach?
Construct a tandem dimers of wild type Aqua1 and C1892 mutant channel and express in xenopus oocytes
DNA sequence of aqua1 and mutant bolted together
Advantage of mixing tetramers
Force each tetramer to contain 2 wt and 2 mutant channels
if you inject both I there it contains lots of different combinations
Experiment method for discovering tetramer shape
- Express AQUA1-AQUA1 or AQUA1-C189S or C189S-C189S dimers in oocytes. The dimers produce functional water channels- effect of mercurial
- Hg reduced the permeability of AQUA1-AQU1 dimers to background levels
- Hg reduced the aqua1-c189s dimers by 50%
4.
Results of tetramer shape experiment
Hg reduced the permeability of AQUA1-AQU1 dimers to background levels- all 4 pores blocked
Hg reduced the aqua1-c189s dimers by 50%
Hg has no effect on water permeability of C189S-C189S
*each 1 of 4 units has an individual water channel- functional water channel
What could the central pore be linked to
Ion transport or gas- multifunctional
Predicted pattern of water movement
Water has H bonding, predict if single file movement protons would piggyback through pore- however this doesn’t allow proton movement- something happens which decouples movement
How was water pore selective?
As water comes through H bonds are broken and form bonds with NPA motif- making pore water selective
Basis of Hg inhibition
Mercury selective- identify where it is sitting
crystal structures with mercury in place and examined- used bacteria aquaporin (usually isn’t mercury selective as it doesn’t contain C189S but add it)
- mercury binds in middle of pore
- signalling pore to prevent water movement
Aquaporin 3
Transport other small molecules
on basolateral membrane of collecting duct and skin
transport urea and glycerol
Look at the action of AQUA3
inject aqua3 into oocyte
inhibited by mercury- Hg sensitive- has cysteine location
- uptake studies-drop into radiolabelled glycerol and urea- left for 15 minutes, taken out, washed out and measured amount of radiolabelled oocyte
Aqua6 antiobody studies show
Colocalisation with H ATPase in intracellular vesicles in a-intercalated cells
unorthodox- not traditional way
Function of AQUA6
Water permeability
acts as a chloride conductance
*interestingly both stimulated by Hg- Add Hg= increase Cl
Where is aqua6 found?
Found in the cell membrane
expressed In the kidney and co-localises with protein pump
How does Ph affect AQUA6 function?
PH sensitive
stimulated by acidic PH- normal ph7.5, changed to 7.4 and there is an increase in H20 permeability and Cl conductance
How does AQUA6 balance out PH
brings protons into vesicel
PH more acidic= activate AQUA6- bring cl into vesicle to balance out charge
Aquaporin family tree
Aquaporins- 0,1,2,4,5
Aqua-glyceroporins- 3, 7,9,10- permeable to water and urea and glycerol
Unorthodox- 6,8,11,12- NPS rather than NPA
*over 900 members of the MIP superfamility have been identified
Links of AQP2 to disease
Diabetes insipidus
- mutations in AQP2- mutation near the channel pore= reduces channel permeability to H20
- Disrupt trafficking of AQP2 to membrane
affects of mutations in aquaporin 2
Unable to concentrate urine
don’t respond to vasopressin
something in water channel prevents pore from functioning
Different types of water transport in epithelia
- Reabsorptive epithelia- proximal tubule= AQP1
- Secretive epithelia- salivary gland= AQP5
- Reabsorptive epithelia- small intestine= No AQP
What is the importance of AQP1 in the proximal tubule
Demonstrates an extremely high water permeability
role in transport of water
KO aqp1= decrease in Pf by 78%
Conditions for AQP1 reabsorption in the proximal tubule
under near isotonic conditions
create conditions where lumen is slightly hypertonic
dilute lumen, osmotic gradient - dive water through AQP1
hypertonic conditions in proximal tubule- how to get them
Na and Cl solute uptake at the apical membrane
forms DF for water reabsorption
Small gradients
Proximal tubule- evidence for iso-osmotic fluid reabsorption 1
Used microperfused proximal tubule segments
- perfusate
- adsorbate
- basolateral outlet= droplet of oil- freshly generated
Results from iso-osmotic fluid reabsorption using oil droplet
looked at proximal convulted tubule and straight tubule
perfusate- osmolarity dropped, generated gradient is smaller
Part 2 of iso-osmotic fluid reabsorption
Looked at the collection duct, capillary and absorption
under high and low flow rates
Collection Is hypertonic in capillary= huge amount of H20 absorption
*micropuncture technique in vivo- exposed kidney of animal, put peptide into capillaries and tubule itself
*solution containing 154nM NaCl= unrealistic conditions
Overall conclusion
Transport by the proximal tubule generates favourable osmotic gradient for H20 reabsorption- extremely high water permeability of the tubule gradient is small and probably due to the generation of hypotonic lumen
Support of the hypothesis of a hypotonic lumen comes from work on the aqua1 KO mouse
WT high aqua1- generate hyertonic lumen, gradient small
KO aqua1= H20 cant move across
- gradient much larger
- much more hypertonic
Salivary gland iso-osmotic fluid secretion
Lumen hypertonic
secreted salt across
- aqua5 drives H20 movement responding to osmotic gradient- salt secreted H20 follows
AQUA5 knock out
Loss of aqua5 prevents the complete dissipation of the lumen hypertonicity
the result is the production of a small volume of saliva with a high salt content
wt= high salt secretion, osmolarity near isotonic- generate osmotic gradient
KO- 50% of saliva produced, high concentrated saliva- limited- H20 movement
aquaproins in the small intestine
Lacks aquaporin
faced with hypertonic luminal environment
typical reabsorptive epithelium
Lumen and blood
Low osmolarity to high- Na Cl solute go to blood
so water follows through a tight junction- through the tight junction
High osmolarity ~500-550mOsm- drive water secretion as well as secretion
Wet transport proteins
Small co transporter also transport water as part of their normal operation
- KCC4- 500
- hSGLT1-235
- GluT2- 40-100
- KCC1- 590 molecules of water- doesn’t transport water=thick ascending limb
Upshot
water can be transported against an osmotic gradient
Exposed cell hypotonic shock solution
cells start to shrink
add 50mmol of KCL- swell- co transport works against 100 gradient, uses KCC4 co transporter up hill
How do they show that KCl transports water?
They block uptake of KCL and there is a cell shrinkage so shows it a co transport
What are the modes of water transport
- AQP1- osmosis
- KCC- co transport
- SGL1, EAAT1-Co-transport + osmosis
microgradients formed
Layer next to cell remove solute from membrane and get slightly hypertonic inside- allows osmosis of water
Implications for water reabsorption- small intestine
Apical- SGT1, GLU2, H20
Basolateral- NA/K ATPase, KCC, GLU2
GLU2- gets inserted into the apical membrane after food, and in basolateral allowing water reabsorption
*Once you’ve had your meal, create conditions in the lumen where you have hypertonic solution
Membranes and gases
Membrane with low gas permeability
aquaporin1 as a gas channel
physiological relevance- RBC and Colton null
modelling of gas transport
What is overtons law?
Permeability of membrane to a solute is proportional to the oil/water partition coefficient for that solute
Gases such as O2 and CO12 have a high solubility in oil so a natural extension of this law was that- all biological membranes were freely permeable to gases
Experiment for overtons law
- take jar with water and oil in it
- put in solute, shake up and measure conc
Highly soluble in oil
membrane permeability, implied that they have freely soluble gases
Challenging the concept in 1990s - gas membrane
- make artificial lipid bilayer- no cholesterol or membrane proteins and has nigh contamination with decone in membrane increase fluidity= increase CO2 permeability
- measure co2 permeability
- generated membrane with limited permeability to gases
Effect of NH4/NH3 on PHi
- NH3- NH3 + H - NH4
- ammonia- weak base, one inside the cell combines with proton- alkaline - NH4- NH4+ - NH3 +H
- ammonium - slower uptake, isn’t permeable across bilayers, ammonium will substitute for K , dissociates to form ammonia and proton- acification
If you have ph 6.1=
Half CO2 and Half bicarbonate
Effect of CO2/ HCO3 on PHi
- CO2- CO2 + H20 - HCO3 +H
- CO2 moves into cell, combines with water and dissociates and proton- gets acidification - HCO3 - Hco3 + H - CO2 + H20
- bicarbonate into cell- alkylation
Thick ascending limb permeability
Apical= high permeability to NH4 but extremely low permeability to NH3- reuptake of ammonium but limited permeability to ammonia Basolateral= high permeability to NH4 and NH3
If you add ammonium Cl to the basolateral side
Alkylation followed by acidification
permeability to ammonia and ammonium
Gastric gland permeability to CO2 and HCO3
Luminal change- 100% CO2
Bath changes- 1% CO2, 6%CO2
Method for Gastric gland permeability to CO2
- open stomach, isolated gastric gland and set up perfusion pipettes
- exposed to bicarbonate and CO2
- exposed to 1% CO2- got expected acidification, basolaterol side normal
- lumen- 100% CO2, take PH down to PK for CO2
Eating meals stomach creates conditions where the PH is close to 1
Acidic
Protects itself and creates tight tough apical membrane preventing stomach digesting itself
What is the basis of the low permeability to the gas?
- experiment using liposomes
2. using MDCK cells
How does cholesterol concentration affect permeability to membrane?
High cholesterol- reduce fluidity of membrane, make tighter. creates an artificial bilayer and changed cholesterol/ lipid content
Low cholesterol- high permeability to CO2, couldn’t reliably measure as so high
increase cholesterol= decrease permeability
What is the basis for low permeability to gases?
Membrane with low cholesterol levels- 30% for example many cancer cell lines, CO2 permeability high enough to support metabolic demands
membrane with high cholesterol levels- 77%- example apical colonic crypt- barrier function for limiting gas transport
RBC and apical membrane on proximal tubule have cholesterol content in 45%- not support level of CO2 transport measured
Effect of CO2 on intracellular PH in xenopus oocyte
10mm HCO3/1.5% CO2
PH drops
Correlation between CO2 and H20
CO2 permeability is proportional to H20 permeability
the more aqp1 channels present the faster the rate of acidification
Effects of PCMBS
organic mercurial
compound binds to cysteine
decreases CO2 and AQP1 CO2 induced acidification rates
Summary of AQ1 expressing in oocytes
Express AQP1 in xenopus oocytes increase CO2 permeability
The increase in CO2 permeability is proportional to the expression level in AQP1
organic mercurial agent PCBS inhibits AQP1 dependent increase in CO2
The C189S mutant increases CO2 permeability to the same degree as the WT
CO2 permeability of RBCs- colton null
Colton null RBC CO2 permeability is greatly reduced compared to normal RBC
Colton null= CO2 permeability is unaltered by PCMBs
CO2 permeability in Colton null and wt is inhibited by DIDs
AQP1 structure
2.2 angstroms by Xray crystallography
AQP1 from bovine RBC
Pores
1. aquapore - hydrophilic and hydrophobic
2. central pore- main hydrophobic gated by hydrophobic reside
Molecular dynamics- stimulation for AQP1
CO2- 6 through the 4 water pores, 4 through central pore
O2- 1 through water pore, 6 through central pore
Effect of cGMP on AQP1
AQP1 closed- no cGMP
AQP1 OPEN- cGMP
db cGMP effect
increase H20
decrease AQP1/5
