Muscarinic Acetylcholine Receptor Biology Essay.
Muscarinic Acetylcholine Receptor Biology Essay.
Using rat brain membranes, buffer, atropine and 3H-QNB you will produce a displacement curve for QNB by atropine, using a filtration method to separate bound QNB from free QNB. Radioactivity on the filters will be measured by scintillation counting and, after correction for counting efficiency, will be converted into molar units from specific radioactivities.Muscarinic Acetylcholine Receptor Biology Essay.
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Introduction:
Receptors for acetylcholine are present in many tissues and can be characterised as falling into two main types, muscarinic or nicotinic, on the basis of their ability to bind muscarine or nicotine respectively. Several substances are known that bind to the muscarinic acetylcholine receptor (mAChR): some of these are agonists (which bind and elicit a response) and some are antagonists (which bind but do not elicit a response). In general, antagonists are used to measure receptor binding as they bind with a higher affinity (lower KD) than agonists bind.
In this experiment you will investigate some of the properties of mAChR in rat brain membranes by means of 3H-quinuclidinyl benzilate (3H-QNB) binding.
This experiment is based upon an article by Yamamura & Snyder (1974) Proc Natl Acad Sci USA 71: 1725-1729 (See course website.)
Requirements:
1. Rat brain membranes – store on ice. (See p for preparation method).
2. Sodium potassium phosphate (NaKP) 50 mM pH 7.4
standard 3H-QNB/NaKP assay mix (NaKP + 1.3nM 3H-QNB,
11.2 x 102 Bq/pmol
high concentration 3H-QNB/NaKP assay mix (NaKP + 6.5 nM 3H-QNB,
11.2 x 102 Bq/pmol
atropine solution (10 μM – MW 290)
* QNB AND ATROPINE ARE TOXIC SO HANDLE WITH CARE *
3. Small glass test tubes, micropipettes – 200 μl (YELLOW TIPS), 1000 μl (BLUE TIPS), 5000 μl (WHITE TIPS)
4. Multiplex filtration apparatus + GF/C glass fibre filters (2.5 cm diam) + forceps
5. Scintillation mini-vials + ‘Ultima Gold’ scintillant
Methods:
All assays have a final volume of 2.0 ml, made up of 1.5 ml 3H-QNB assay mix, 0.3 ml water or atropine. The assay is started by adding 0.2 ml membranes. The excess atropine added to the controls displaces the specific and saturable (i.e. receptor-bound) QNB leaving the non-specific, non-saturable QNB bound to the membranes.
The assays are left for the appropriate length of time, stopped by adding 2.0 ml NaKP to increase the volume and filtering immediately through glass fibre filters. These are washed with NaKP and counted overnight in a scintillation counter.
Day 1
1. Make up two ‘bulk’ assays, one to measure total QNB binding (with water) and one to measure non-specific binding (with atropine). Set up two 50 ml conical flasks thus:
A
B
3H-QNB (1.3 nM)
30.0 ml
30.0 ml
water
6.0 ml
0.0 ml
atropine
0.0 ml
6.0 ml
(this is enough for 20 assays – you will do 18 assays)
2. Set up a filter tower with six GF/C filters. When you are ready, quickly add 4.0 ml swirled membranes to each flask and swirl to mix.
3. Now remove 2.0 ml aliquots to filters, three for each flask, making sure that you know which are from flask A and which from B.
*USE SEPARATE PIPETTE TIPS FOR FLASKS A AND B*
Note that if you contaminate the QNB solutions with atropine
it will completely abolish all binding
Filter quickly through fresh GF/C filters.
4. Wash each filter with 5 ml NaKP, remove filters to mini-vials, add 5 ml scintillant, invert, leave at least 1 hr, invert again and count the radioactivity in the scintillation counter.
5. Repeat steps 3 & 4 at times =10, 20, 30, 45 and 60 mins.
6. Using the swabs provided, take six separate samples to check for radioactive contamination, for example by rubbing gloves, bench or anything that might have been in contact with 3H-QNB. Carefully note the origin of each swab. Then put each swab into a separate vial containing 5 ml of scintillant, as before, record the treatment of each, and send them for counting. This is a standard safety procedure when dealing with radioactive chemicals. The amounts of tritium involved in this experiment are unlikely to damage your health. Nevertheless this is a useful exercise to find test your technique before you make a mistake with 32P or 125I (much more damaging).Muscarinic Acetylcholine Receptor Biology Essay.
Day 2
Note that you need to take great care to get the correct volumes of each solution into the appropriate tubes. The more care you take, the better will be your results
Determine IC50 for atropine (i.e. that atropine concentration which displaces 50% of QNB binding).
Take 5 small glass test tubes (1-5) and put 1200 μl of distilled water in each.
Now add 300 μl of 10 μM atropine to Tube 1, mix well and transfer 300 μl to Tube 2.
Mix well and transfer 300 μl to Tube 3.
Repeat up to Tube 5.
Calculate the atropine concentration in each tube.
Set up 7 triplicate glass tubes (A1, A2, A3, B1 …… G3) as follows:
Tubes
300ml of
1.3 nM QNB assay mix
A
10mM atropine
1500ml
B
Tube 1
1500ml
C
Tube 2
1500ml
D
Tube 3
1500ml
E
Tube 4
1500ml
F
Tube 5
1500ml
G
distilled water
1500ml
As rapidly as possible add 200ml membranes to each tube. Proceed as described in 2)….4) above, using the incubation time you calculated from Day1’s experiment (it should be at least 45 minutes). It is best to start the reactions in two batches, with 5 minutes between each batch to allow you time to filter the first batch before the second batch is due.Muscarinic Acetylcholine Receptor Biology Essay.
Calculate the average radioactivity bound to each triplicate set of filters and convert this value into suitable units of QNB bound (nanomoles or picomoles). Plot these values against log10[atropine]. Estimate the IC50 from the midpoint of the curve and compare your result with that obtained by Yamamura & Snyder.
While you are waiting for the reactions to reach equilibrium, carry out a Lowry assay for protein (see p) so that you can calculate specific QNB binding in fmol QNB per mg protein, and compare your value to that given in the Yamamura & Snyder paper.
You will be told in the class what quantities of membrane preparation to use in this assay.
Day 3
Note that you need to take great care to get the correct volumes of each solution into the appropriate tubes. The more care you take, the better will be your results
Determine KD for QNB. You will make lower concentrations of QNB by diluting the standard QNB assay mix with NaKP; higher concentrations can be made from the high concentration 3H-QNB mix but this is strictly limited at 20 assays per group – don’t waste it.
Label eight test tubes 1-8.
Tube
1.3 nM QNB mix
6.5 nM QNB mix
NaKP
ml
ml
Ml
1
0
7.50
0.00
2
0
2.50
5.00
3
0
5.00
2.50
4
0
3.20
4.30
5
6.00
0.00
0.00
6
2.50
0.00
5.00
7
5.00
0.00
2.50
8
3.50
0.00
4.00
Label eight sets of triplicate tubes A1, A2, A3….H3. Add the water or atropine last.
Tubes
1500 μl from Tube #
300 μl
A
1
Water
B
2
Water
C
3
Water
D
4
Water
E
5
Water
F
6
Water
G
7
Water
H
8
Water
Now label a separate set of eight tubes label A4, B4…H4. Set these up as the previous but add Atropine instead of water. Note that this set is not done in triplicate.
Add 200 μl of membrane preparation to each tube. Incubate the tubes as described in 2)…4) above, the incubation time being that determined on Day 1. It is best to start the reactions in two batches with 5 minutes between to allow you time to filter the first batch before the second batch is due.
Calculate the average radioactivity bound to each triplicate set of filters and convert it into amounts of QNB (nano- or picomoles). Draw a straight line through the atropine controls, and subtract the values for each real or ‘estimated’ atropine control from the ‘water’ values and use these data to calculate the bound and free QNB values.
While you are waiting for the reactions to reach equilibrium, carry out a Lowry assay for protein (p) so that you can calculate specific QNB binding in fmol QNB per mg protein, and compare your value to that given in the Yamamura & Snyder paper.
The data from this experiment may be analysed by Scatchard analysis. This will be discussed during the following session. Further information about this and other methods of analysis can be found at:
http://www.curvefit.com/introduction75.htm
Dispose of your radioactive equipment and toxic chemicals in the correct places.
Data analysis
Questions to think about:
How many dpm should be present in each assay? (Calculate this.)
What is the likely nature of the non-specific binding?
Comment on the rate of binding for the specific and the non-specific binding.
What other methods are available for measuring receptor-ligand equilibria?
If the off-rate were fast (e.g. half-life of around 1 second) what method of assaying the receptor-ligand binding might be suitable?
Does the QNB concentration affect the IC50 of atropine?
LOWRY ASSAY FOR PROTEIN
Reagent 1:
0.5 ml copper tartrate has been mixed with 50 ml alkaline carbonate on the day of use.
copper tartrate (0.1 g CuSO4.5H2O added to 0.2 g NaK tartrate in 20 ml water)
alkaline carbonate (2 g NaOH in 20 ml water and adding 10 g Na2CO3, made up to 100 ml with water)
Reagent 2:
Commercial Folin-Ciocalteau reagent 1:1 in water
Method:
In a series of test tubes, add the volume of membrane announced at the start of the class and make this up to 1 ml with water.
Prepare tubes containing 0, 50, 100 150 and 200 μg bovine serum albumin (BSA) made up to 1 ml water. The concentration of BSA you are supplied with is 1 mg.ml-1.
Add 1.5 ml Reagent 1.
Mix well and leave to stand for 10 min at room temperature.
Add 0.3 ml Reagent 2, mix well and leave for 30 min.
Read at 660 nm.
Plot the data from the standard BSA tubes and calculate the protein concentration in the membranes.
PREPARATION OF RAT BRAIN TISSUES
Rat brain membranes for QNB binding experiment
Rat brains were homogenised in 10 volumes ice-cold 0.32 M sucrose/0.1 mM PMSF with a Teflon-glass Potter homogeniser. This was centrifuged at 12000g x 10 minutes and the pellet resuspended in original volume of sucrose and frozen in aliquots.Muscarinic Acetylcholine Receptor Biology Essay.
