Lectures 5&6: Cholinergic receptors Flashcards
What are the two families of Cholinergic/acetylcholine receptors?
Nicotinic receptors (ionotropic)
Muscarinic (metabotropic)
Explain features of nicotinic receptors
type of cholinergic receptors
ionotropic
Illicit a response through allowing passage of ions across membrane
(symp + parasympathetic)
Fast (μs/ms)
Explain features of Muscarinic receptors
type of cholinergic receptor
metabotropic
G-protein receptor
slower response due to signalling events
parasymathetic
slow (ms/sec)
What are common structural features of nAChR subunits
4 transmembrane domains (α-helical) (3+1 motif)
Extracellular domain (β-sheet)
Extracellular disulphide bond
Explain the use of electric rays in purification of receptors
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)
Explain what Edman degradation (old technique) has been replaced by
Mass spectrometry
Explain how a partial AA sequence can lead to a full sequence discovery
Screen partial sequence in cDNA libraries
Can then sequence positive clones and predict primary structure
What can be learnt from finding out a full AA sequence of a protein/receptor?
- Homology with other receptos/subunits
- post-translational modifications
- Use a Hydropathy plot to get an idea about topology (e.g. hydrophillic/phobic)
Explain why (apart from abundant proptens e.g. rhodopsin) expression in another model for membrane proteins is needed
Most receptors are found at very low concentrations
Give examples of different membrane protein expression systems
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)
Explain heterologous expression of receptors in Xenopus oocytes
Provides a valuble tool to understand at a functional level how nAChR and other receptors work
Explain limitations of eukaryotic membrane protein expression in E.coli
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
Explain extraction of membrane proteins
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)
explain different seperation techniques for membrane proteins
- 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
Explain how membrane proteins are reconstructed into lipid vesicles
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
Chemical labelling + identification of ligand binding sites
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)
Identification of the ACh binding site on the nAChR
(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
Explain how the structural characterisation of nAChR occurred
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
Structure of nAChR
60 angstrom protrusion from the surface of the bilayer which is composed of beta sheets – contains ligand binding domain
Transmembrane domains to transport ions
Structure of the synaptic domain of nAChR
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
TMD structure of nAChR
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
Explain the agonist binding site of nAChR
Main binding site on α-subunit with residues contributed from a secondcomplementary subunit (either δ or ɣ)
Explain the pharmacophores for ACh binding
Choline head group with positive charge
Ester group with hydrogen bonds
How can the importance of ACh’s pharmacophores be highlighted?
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)
What is the binding of Ach to receptors binding pockets driven by?
NOT charge-charge interaction
actually cation pi intercations
Explain a model for the Ach binding pocket
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
explain features and challenges of studying Acetylcholine Binding Proteins (BPs)
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
Explain roughly the structures of angosists and antagonists of AChBP
Antagonists:
Methyllcaconitine
a-Conotoxin lml - 12 AAs in length
Agonists:
Lobeline
Epibatidine (small)
Explain agonist and antagonist binding to AChBP
Agonist - results in folding of the C loop over the binding pocket
Antagonist - C-loop pushed away from the binding site
Explain an experiment that showed how agonist binding to AChBPs lead to channel opening
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
Explain the movement of the different TMDs in Ach binding to AcHBPs
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
Explain the role leucines play in occluding the passage of AchPBs (in urwins model)
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
Explain how from Urwins expeiments, what Ach binding model we use
- ACh binds
- C-loop 9part of beta sandwich wraps around ligand
- Initiates rotation of b-sandwich
- Movement of Cys and b1-b2 loops
- Release of M2-TMD from locked state
- Rotation of a-subunits
- Unlocking of the hydrophobic gate
- Passage of ions
What made Urwins model be questioned?
Development of crystallography - Identified two ligand gated ion channels found in bacteria
What were to two ligand gated ion channels found in bacteria identified by crystallography (NOT ACh receptors)
GLIC and ELIC
Explain how GLIC and ELIC were analysed using crystallography and differences from channels derived from EM
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)
What does ‘hole’ analysis tell us about GLIC and ELIC ?
allows calculation of size of the channel passing through the bilayer
Explain the similar gating mechanisms seen in ELIC and GLIC and urwins mechanism
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
What was different about the gating mechanisms observed in ELIC and GLIC compared to urwins mechanism?
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
Whats important to take into account when comparing urwins therory with the ELIC/GLIC data
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
Features of Muscarinic Acetylcholine Receptors (mAChR)
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
agonists and antagonists of mAChR
Agonists:
Acetylcholine
Muscarine
Antagonists:
Atropine
Explain the cloning of the mAChR
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)
What is unusual about the mAChR topology
TMD conserved between subtypes (due to this being where ligand binding site is)
Structure of mAChR
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
Explain the different G coupling fo mAChR subtypes
M1R, M3R, M5R - couple Gq/11
M2R, M4R - couple Gi/o
Explain the cascade of mAChR activation
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
explain what the different effectors (M1-5) stumulate within the cell
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
Explain necessary features if the Ach binding site on mAChR (simple)
Ester necessary for high affinity
Hydrophobic surface drives binding
H-bonding necessary
Explain further features of the ACh binding site of mAChR (example M3)
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
Explain insight into subtype selectivity of the mAChR by X-ray structures
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
Do M2 and M3 mAChR share the same molecular scaffold?
YES
What is the molecular scaffold for both M2 and M3
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)
Explain differences in M2 and M3 mAChR arrangement
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
explain the advantage of molecular dynamics (MD) over crystallograpy for understanding mAChRs
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
Explain what happened when Molecular dynamics revealed an extracellular vestibule on the mAChR
Compounds bind to vestibule, previously identified as a allosteric regulator (weaker binding site established in the entry to the helical bundle)
Explain how functional assays can be conducted in Xenopus oocytes for nAChR
-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
What does studies into nAChR using functional assays on Xenopus oocytes facilitate?
- 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
