Lectures 5&6: Cholinergic receptors

Question 1

What are the two families of Cholinergic/acetylcholine receptors?

Answer 1

Nicotinic receptors (ionotropic)
Muscarinic (metabotropic)

Question 2

Explain features of nicotinic receptors

Answer 2

type of cholinergic receptors
ionotropic
Illicit a response through allowing passage of ions across membrane
(symp + parasympathetic)
Fast (μs/ms)

Question 3

Explain features of Muscarinic receptors

Answer 3

type of cholinergic receptor
metabotropic
G-protein receptor
slower response due to signalling events
parasymathetic
slow (ms/sec)

Question 4

What are common structural features of nAChR subunits

Answer 4

4 transmembrane domains (α-helical) (3+1 motif)

Extracellular domain (β-sheet)

Extracellular disulphide bond

Question 5

Explain the use of electric rays in purification of receptors

Answer 5

Homogenise membranes

due to being so rich in nicotinic acetylcholine receptor, can separate membranes by centrifugation

Harvest at boundary, and run an SDS page (bands on gel in different intensities)

Question 6

Explain what Edman degradation (old technique) has been replaced by

Answer 6

Mass spectrometry

Question 7

Explain how a partial AA sequence can lead to a full sequence discovery

Answer 7

Screen partial sequence in cDNA libraries

Can then sequence positive clones and predict primary structure

Question 8

What can be learnt from finding out a full AA sequence of a protein/receptor?

Answer 8

• Homology with other receptos/subunits
• post-translational modifications
• Use a Hydropathy plot to get an idea about topology (e.g. hydrophillic/phobic)

Question 9

Explain why (apart from abundant proptens e.g. rhodopsin) expression in another model for membrane proteins is needed

Answer 9

Most receptors are found at very low concentrations

Question 10

Give examples of different membrane protein expression systems

Answer 10

Xenopus (bigger than bacteria) makes them goof for functional assays

E.coli (can grow in large quantities and cheap)

Insect cell culture (small eukaryotic)

Tissue Culture (price is more, increased significantly) (has ER which is advantage as more network of membranes)

Question 11

Explain heterologous expression of receptors in Xenopus oocytes

Answer 11

Provides a valuble tool to understand at a functional level how nAChR and other receptors work

Question 12

Explain limitations of eukaryotic membrane protein expression in E.coli

Answer 12

Different codon usage in bacteria

Limited membranes (no Enodplasmic Reticulum)

Different membrane protein insertion machinery compared to eukaryotes

No post-translational modifications

Lipid composition differs from eukaryotes

Over-expression of heterologous protein may lead to inclusion bodies.

Multi-subunit proteins difficult to assemble

Question 13

Explain extraction of membrane proteins

Answer 13

Traditional detergents: ionic (e.g. SDS), Zwitterionic (e.g. CHAPS), non-ionic (e.g. DDM)

Modern approaches: amphipoles to make nanodiscs e.g. SMALPs e.g. surfactants, disrupt membrane and disrupt small patches of membrane, at points where receptor is (retain membrane integrity)

Question 14

explain different seperation techniques for membrane proteins

Answer 14

• If cloned, tag based affinity chromatography

His10/12 (Higher column affinity than His6) Doenside – mrmbrane receptors have less accesability

Maltose Binding Protein (also useful for targeting) - 2 benefits, can be tag for affinity purification 2nd RW

• Ligand Affinity Chromatography (alows to isolate only receptors we want)

α-bungarotoxin (AChR)
neurotensin (neurotensin receptor) (functional receptors will bond to column, non-functional will not)

• Functional receptor – able to transduce a signal

Combination of the above

Crude purification with tag based system

Functional selection with agonist based column

Question 15

Explain how membrane proteins are reconstructed into lipid vesicles

Answer 15

Detergents exist in equilibrium between micelle and monomer

Slow removal of monomeric detergent in presence of solubilized lipid allows formation of proteoliposomes

Common methods:

• Dialysis
• Biobeads
• Dilution

Downside of dilution, dilutions have to use are very large, so usually done by dialysis or biobeads

Can think about doing transport assays and electrophysiology

Question 16

Chemical labelling + identification of ligand binding sites

Answer 16

Early work - focused on chemical labelling:
take native agonist - modify in a way to allow tagging of receptor binding sites, done in a few ways:

Bromoacetylcholine - contains bromine group that will react with a free cystine

Acetylcholine mustard - has 3 membered ring, very reactive form, will react with residues in close proximity - the quaternary ammonium group binds to the ligand binding site

DDF - Amine that binds to ligand binding site (and a reactive functionality that binds to residues nearby)

MBTA - quaternary ammonium group to target ligand binding site + reactive functionality that binds residues nearby

For all of these, introduce tritium or C14 into ligands, then react with receptor of interest

Can do a coomassie gel - 5 different subunits - isolate the alpha, and undergone a proteolytic digest (cleave with trypsin), get a pattern, then look at where the radioactivity is on the gel using gel radiography - find where radio-isotopes were binding

THEN - can do edman degradation sequencing - this time sequence protein of interest but as each AA is released, you monitor for radioactivity (therefore know which residue was labelled with labelling agent)

Question 17

Identification of the ACh binding site on the nAChR

Answer 17

(Pedersen 1986)
From chemical labelling and edman degradation sequencing, can start to build up a picture of which residues are in the binding site ect.

• No negative charged residues - site rich in tryptophans and tyrosines (therefore rich in aromatic residues)

-located close to disulphide. bond Cys192/193

• although most of the labelling was on alpha subunit, some labelling on others (gamma/delta) - demonstrated binding sites at interface between two subunits

Question 18

Explain how the structural characterisation of nAChR occurred

Answer 18

Took torpedo and did Cryo-electron microscopy of nAChR helical arrays:

Initially: not high quality images

Realized of he left samples at –80 for two years, started to form these tubular arrays

Pattern seen is a radial distribution of acetylcholine receptors, can use this pattern to get high resolution electron microscopy data

Then electron diffraction done:

Using a conventional electron microscope, but instead of refocusing the electrons underneath the specimen what you do is collect the diffraction pattern

The diffraction pattern contains information for all the receptors that have been irradiated, use this to reconstruct

Question 19

Structure of nAChR

Answer 19

60 angstrom protrusion from the surface of the bilayer which is composed of beta sheets – contains ligand binding domain

Transmembrane domains to transport ions

Question 20

Structure of the synaptic domain of nAChR

Answer 20

Beta sandwich – two beta sheets packed on top of one another – then a C loop (involved in agonist binding)

end of beta strands - loop regions, which interact with TMD

Relatively rigid structures - easy for a conformational change to be propagated to the level of the lipid

Question 21

TMD structure of nAChR

Answer 21

5 subunits –alpha, gamma, alpha, beta, delta

Arranged symetricaly around channel that’s forming in the centre

Channel is composed of M2 TMDs, second TMD that’s lining the channel where the ions will transport through

Outside this second TMD - region composed of the 1st and 3rd TMD

And then out on the periphery – M4 TMD

Question 22

Explain the agonist binding site of nAChR

Answer 22

Main binding site on α-subunit with residues contributed from a secondcomplementary subunit (either δ or ɣ)

Question 23

Explain the pharmacophores for ACh binding

Answer 23

Choline head group with positive charge

Ester group with hydrogen bonds

Question 24

How can the importance of ACh’s pharmacophores be highlighted?

Answer 24

Looking at inhibators of the Ach receptor:
Strychnine - separation between quaternary ammonium group and one of the hydrogen receptors is 4.97A, comparing this to the bound acetylcholine, values very close (5.09A)

Question 25

What is the binding of Ach to receptors binding pockets driven by?

Answer 25

NOT charge-charge interaction
actually cation pi intercations

Question 26

Explain a model for the Ach binding pocket

Answer 26

Structure of bound ACh determined from solid-state NMR

Docking to AChBP reveals

• Extensive cation-π interactions
• Binding of positive charge qaternary group

Modelled with AChBP

• Leu in close proximity to CO of ACh
• Homologous to α7 (neuronal receptor) not α2βγδ (receptor in Torpedo nobiliana) – lower affinity

C-loop folds over bound ligand

Question 27

explain features and challenges of studying Acetylcholine Binding Proteins (BPs)

Answer 27

Membrane proteins: challenging to crystalize (lower resolution)

Can get the AcHBP to 2A resolution

Soluble AChBP: Homologue from snail

Important to thin in pharmacology about the importance of size of dug studying e.g. Ach stretched out is 7A, so If resolution is only 3 then that means its difficult to work put where the pharmacaphores are

So need high structural resolution information

Question 28

Explain roughly the structures of angosists and antagonists of AChBP

Answer 28

Antagonists:
Methyllcaconitine
a-Conotoxin lml - 12 AAs in length

Agonists:
Lobeline
Epibatidine (small)

Question 29

Explain agonist and antagonist binding to AChBP

Answer 29

Agonist - results in folding of the C loop over the binding pocket

Antagonist - C-loop pushed away from the binding site

Question 30

Explain an experiment that showed how agonist binding to AChBPs lead to channel opening

Answer 30

have togo back and look at overall conformation of Ach Receptor
Urwin 2005

Tried to trap open conformation of the AChR, did this by:

Take electron microscopy grids and dropped them

Dropping them causes acceleration at 9.81m/s2 (gravity)

Work out how long it takes it to fall into a bath of liquid ethane where it will freeze, to do this he:

Took at atomiser (like whsts used for perfume) and put Ach inside. Dropped grid, sprayed with atomiser and as it fell through he adjusted the distance at whucgh the liquid ethane was so that he got it just at the point of the open receptor

What he saw:

As the Ach was binding, rotation of different subunits
Rotation of C loop causes movement of Cyc Loop and b1/b2 loop, pulling them away from the transmembrane domain

These two loops are important in channel gating

Question 31

Explain the movement of the different TMDs in Ach binding to AcHBPs

Answer 31

When Ach binds, C loop reaches around over the agonist, movement is propagated down the M2/M3 level, and 2 loop regions get pulled out the TMD

Movement of β1-β2 and Cys loop unlocks the M2 helix

Question 32

Explain the role leucines play in occluding the passage of AchPBs (in urwins model)

Answer 32

ring of leucine residues that occlude the passage, so we cannot get the passage of ions through the lipid bilayer

When Ach binds, C loop wraps round, and displacement fo transmembrane domains, we see rotation of the leucine side chains away from the central pool, ions can now pass through bilayer

Question 33

Explain how from Urwins expeiments, what Ach binding model we use

Answer 33

1. ACh binds
2. C-loop 9part of beta sandwich wraps around ligand
3. Initiates rotation of b-sandwich
4. Movement of Cys and b1-b2 loops
5. Release of M2-TMD from locked state
6. Rotation of a-subunits
7. Unlocking of the hydrophobic gate
8. Passage of ions

Question 34

What made Urwins model be questioned?

Answer 34

Development of crystallography - Identified two ligand gated ion channels found in bacteria

Question 35

What were to two ligand gated ion channels found in bacteria identified by crystallography (NOT ACh receptors)

Answer 35

GLIC and ELIC

Question 36

Explain how GLIC and ELIC were analysed using crystallography and differences from channels derived from EM

Answer 36

Crystalised in open and closed conformational states

Now have high resolution crystal structures of both states

Very similar to ones derived from EM – difference is slightly more squeezed together (could be a result of the crystal packing)

Question 37

What does ‘hole’ analysis tell us about GLIC and ELIC ?

Answer 37

allows calculation of size of the channel passing through the bilayer

Question 38

Explain the similar gating mechanisms seen in ELIC and GLIC and urwins mechanism

Answer 38

Displacement of C-loop

Displacement of b1/b2 loop (Cys not as important in gating mechanism)

See the same conformational change in the beta sandwich

Question 39

What was different about the gating mechanisms observed in ELIC and GLIC compared to urwins mechanism?

Answer 39

Instead of rotational events that erwin was suggesting, more of an opening of he two end TMDs (like a pair of scissiors) - Helices 2 and 3 swing open by about 12° opening the channel

Question 40

Whats important to take into account when comparing urwins therory with the ELIC/GLIC data

Answer 40

neither model may be correct, one may be or the other, note that this is a bacterial system so may not necessarily reflect what’s going on, although high resolution data is a lot easier to interpret correctly

Question 41

Features of Muscarinic Acetylcholine Receptors (mAChR)

Answer 41

Classical G-protein coupled receptor (GPCR) - 7 transmembrane domains, 4+3 topology in alpha helices packing together, ligand binding site

Mediate signaling by G-proteins:

Coupled to Gi/Go or Gq/G11

Question 42

agonists and antagonists of mAChR

Answer 42

Agonists:
Acetylcholine
Muscarine

Antagonists:
Atropine

Question 43

Explain the cloning of the mAChR

Answer 43

M1 and M2 subtypes first cloned from pigs

Human equivalents identified by low stringency screening of human cDNA libraries, identified two types of muscarinic receptors:

M1 located primarily in the cerebral cortex

M2 expressed primarily in cardiac tissue

Homology screening with hamster β2-adrenergic receptor (more of that next lecture) reveals a further 3 subtypes (M3-5)

Subtypes exhibit different affinities for agonists, antagonists, locations and actions (different pharmacologies)

Question 44

What is unusual about the mAChR topology

Answer 44

TMD conserved between subtypes (due to this being where ligand binding site is)

Question 45

Structure of mAChR

Answer 45

7 transmembrane domains

Extracellular – small loops

Intracellular – large loops (common in GPCRs due to these being involved in interactions with the G-protein)

Desensitization can occur – when you get phosphorylation of C terminal domains – particularly IL3 (intracellular loop 3), this inhibits binding of G-protein, this is one way to modulate these interactions

Question 46

Explain the different G coupling fo mAChR subtypes

Answer 46

M1R, M3R, M5R - couple Gq/11

M2R, M4R - couple Gi/o

Question 47

Explain the cascade of mAChR activation

Answer 47

Molecule of Ach – binds to integral membrane proteins of the 7 transmembrane domains

Acts like a GTP exchange factor

Binds to G-proteins on inside of the cell

This stimulates exchange of GDP for GTP

Causes dissociation of the alpha subunit from the beta gamma subunit

Dissociation means that all three are free to go off and interact with effector systems they are paired with

Question 48

explain what the different effectors (M1-5) stumulate within the cell

Answer 48

M1,3,5:
Increase in PLCβ, [Ca2+], MAP kinases
Decrease in M current

M2,4:
Increase in MAP kinase, GIRK channels
Decrease in Adenyl cyclase, V gated Ca2+ channels

Question 49

Explain necessary features if the Ach binding site on mAChR (simple)

Answer 49

Ester necessary for high affinity

Hydrophobic surface drives binding

H-bonding necessary

Question 50

Explain further features of the ACh binding site of mAChR (example M3)

Answer 50

Key residue – D147 – presents a carboxylate to the quaternary ammonium group

Tyrosines with hydroxyl group begin to form aromatic cages

Ionic interactions and aromatic cage drive binding of the quaternary ammonium group.

High affinity binding (as for the nAChR) requires H-bonding to the ester moiety

Hydrophobic surfaces interact between CH tubes and protein cavity - to support binding

Question 51

Explain insight into subtype selectivity of the mAChR by X-ray structures

Answer 51

Crystal structures obtained for rat M2 and human M3 subtypes

Crystallized in the presence of the antagonists QNB or Tiotropium

Two subtypes couple different G-proteins, potential to provide a structural understanding of G-protein discrimination

Can drive development of subtype selective drugs

Question 52

Do M2 and M3 mAChR share the same molecular scaffold?

Answer 52

YES

Question 53

What is the molecular scaffold for both M2 and M3

Answer 53

7 transmembrane domains (3+4 bundle)

8th amphipathic helix – lying on bilayer, increases amount of protein available on the intracellular surface of the membrane

Large intracellular loops less well defined, challenge to understand G-protein coupling. (also difficult as these are highly dynamic, which is important in their function and to get a crystal structure, we stabalise them)

Question 54

Explain differences in M2 and M3 mAChR arrangement

Answer 54

M3 has a larger binding pocket/site - means a selective drug for this could be made that would be too big to fit into 2 (maybe extend ring structure)

Looking at backbone of ligands (QNB and Tiotropium)– spatial distribution is very similar

Question 55

explain the advantage of molecular dynamics (MD) over crystallograpy for understanding mAChRs

Answer 55

By combining crystallography with MD, means that before complexes formed had to be relatively stable, but now not as much when using MD

Extracellular vestibule was also revealed

also Predict where molecules or drugs are going to move

Molecular dynamics trajectories can also give energy profiles – these showed that the activation barrier (activation energy) for entry into M3 higher (lower for M2)

Provides kinetic selectivity for drug binding and disassociation

Question 56

Explain what happened when Molecular dynamics revealed an extracellular vestibule on the mAChR

Answer 56

Compounds bind to vestibule, previously identified as a allosteric regulator (weaker binding site established in the entry to the helical bundle)

Question 57

Explain how functional assays can be conducted in Xenopus oocytes for nAChR

Answer 57

-Its relavively large meaning its immunable to microinjection and electrophysiology - means good for functional assays

• Inject 4 mRNAs (corresponding to a2,b,y,delta subunits) into oocyte - this gives rise to a functional expression of the receptor
• Assay pharmacology (e.g. Ach binding)
• Follow the activity of receptors using electrophysiology
• Check specificity (e.g. 125I-bungarotoxin binding) - this is an alpha-neurotoxin snake venom, shares a three finger toxin tertiary structure

Question 58

What does studies into nAChR using functional assays on Xenopus oocytes facilitate?

Answer 58

• formation of other subunits/undertanding e.g. in the brain A7 subtype is expressed (made up of 5alpha subunits),can mix an match sunbunits to try understand pharmacology
• site directed mutagenesis - generate mRNA that has a mutation where you want to see if somethings important, inject into Xenopus oocyte, see effect this has on binding of various drugs
• Scale this up for ‘High throughput’ testing- there are now systems that automatically inject, express and follow the electrophysiology