Midterm 2 Content - nAChRs and AChBP Flashcards
What is the structure of a nAchR agonist typically like? What are nAchR agonists/partial agonists/antagonists?
Quaternary ammoniums (N-R4+) with a positive charge.
Full agonists: nicotine, Ach, Epibatidine, Carbacholamine
Partial agonists: lobeline
Antagonists: MLA (Methylylcaconitine) and Alpha-conotoxin-IMI.
What are the different types of nAchRs and what subunits can they consist of?
nAChRs are pentameric cys loops
Muscle nAchRs: 2 alpha, 1 beta, 1 delta, and 1 gamma/epsilon.
- The subunit between alpha must be gamma/epsilon.
Neuronal nAchRs: 3 categories of subunits
- Alpha2-Alpha 6: bind with beta2-beta4 subunits to make heteropentamers
- Alpha7-Alpha10: form homodimers.
What are the 3 domains of an nAchR?
Extracellular ligand-binding domain
Transmembrane domain
Cytoplasmic/Intracellular domain
What is the membrane topology of a nAChR like?
See notes for drawing
n = 5
4 transmembrane regions
M2 is pore-lining
What are the binding sites of muscle nAchRs like?
Between alpha-gamma/epsilon inferface and alpha-delta interface
Binding sites have A, B, C, D, E, and F loops.
What are the binding sites of neuronal nAchRs like?
Homopentamers: 5 binding sites, at every subunit interface.
Heteropentamers: 2 binding sites, each between an alpha and beta subunit.
What kind of receptor did Zhong et al. look at?
Muscle nAChR
How did Zhong et al make unnatural amino acids?
Used nonsense codon suppression, in which a stop codon is put at the site of interest in the gene for the protein.
A tRNA is made with the stop codon anticodon and the unnatural amino acid attached.
Both are injected into an oocyte and expressed.
How do different side chains have an effect on cation-pi binding?
The addition of more electronegative residues pulls electrons away from the aromatic “electron cloud” and the binding energy of the cation-pi bond decreases.
What is EC50?
Effective concentration 50%: the concentration of agonist at which 50% of the maximal response is received.
What is the relationship between EC50 and cation-pi bonding between Ach and nAChRs? Is this changed with nicotine?
Ach
–> For both muscle and neuronal nAChRs: as EC50 increases, cation-pi energy bonding decreases.
Nicotine
- -> the relationship is only maintained with neuronal nAChRs; not muscle receptors.
- -> however, if G153 of muscle nAChRs is mutated to a lysine (K), characteristic of alpha-4 subunits, there are cation pi interactions between nicotine and the receptor.
What residue did Zhong et al discover made the only cation-pi interaction with Ach?
Trp149 (W149) on the B loop of the binding pocket (TrpB)
What did Zhong et al. observe when they tethered a quaternary amine to TrpB?
This resulted in a constitutively active nicotine current.
What receptor did Xiu et al. study?
Alpha4beta2 neuronal nicotinic receptors.
What 4 principal loops define nAChR binding sites? Describe the structure of the binding site. What are 2 major difference between muscle and neuronal nAChR binding sites?
A, B, C, and D loops.
The binding site is like an aromatic box, with a tyrosine at the top and tryptophan at the bottom.
1) G153, located outside the aromatic box on loop C, is only found on muscle nAChRs.
2) The C terminus of Trp B participates with hydrogen bonding with nicotine.
What did Xiu et al observe in muscle nAChRs or neuronal nAchRs upon performing amide to ester backbone mutations on nicotine? What about in Ach?
Amide to ester backbone mutation reduces hydrogen bonding by reducing electronegativity of the carbonyl ozygen.
Muscle nAChR: EC50 did not change in Ach or nicotine.
Neuronal nAChR: EC50 increased for nicotine 19-fold; did not change for Ach.
–> This is because only nicotine hydrogen bonds with the C terminus of TrpB of neuronal nAChRs.
What is AchBP? What organism is it obtained from and what does it resemble?
A soluble protein obtained from pond snail Lymnaea stagnalis.
Closely related to alpha subunits, especially alpha-7 subunit; acts as homolog to ligand-binding domain.
Where is AchBP found naturally in the body?
Released in an Ach-dependent manner into the synaptic cleft by some glia to modulate synaptic transmission?
What is alpha bungarotoxin?
An AchBP antagonist; neurotoxin found in snakes.
What did Brejc study?
The AchBP of Lymnaea stagnalis
What is the structure of AchBP like? What does it resemble?
Homopentamer, consisting of 5 protomers, with no transmembrane or cytosolic domains.
As a principal (inner) and complementary (outer) side, each with loops.
Resembles a “windmill toy”
What does an AChBP protomer consist of?
N-terminal alpha helix
2 310 helices
10 beta strands (a beta sandwich)
What is a Cys loop made of?
A loop consisting of highly conserved amino acids between two cysteine residues, which form a disulphide bond.
What is the main immunogenic region (MIR)?
A region of muscle nAChRs that acts as an epitope in Myasthenia Gravis.
What is the double Cys loop?
Another name for the C loop of nAchRs/AchBP; “sticks out” from the AchBP protomer.
What is Myasthenia Gravis? What are symptoms and treatments?
Autoimmune disease targeting muscle nAChRs at the main immunogenic region.
Results in weakening of skeletal muscle; symptoms include weak muscles, droopy eyelids.
Treatments:
- Neostigmine: acetylcholineresterase inhibitor.
- Prednisone: steroid that relieves muscle weakness.
What is the structure of the AChBP ligand-binding site like?
Consists of a series of loops from the principal (+/A) side of one subunit and the complementary (-/B) face of an adjacent subunit.
Prinicpal (+/A) side: Loop A, Loop B, and Loop C (double Cys loop)
Complementary (-/B) side: Loop D, Loop E, and Loop F.
The ligand-binding site is arranged in an aromatic box with aromatic residues on the floor and hydrophobic side walls.
What is HEPES?
A buffer molecule that contains a positively-charged quaternary ammonium group.
Is larger than Ach and has lower affinity at low concentrations.
Brejc et al. visualized HEPES in the AChBP ligand-binding site.
How did Brejc et al. correct Zhong et al’s model of the muscle nAChR stoichiometry?
The interfaces were correct, but the ordering of the subunits was different.
In a clockwise order, Brejc determined that the order must be..
beta-delta-binding site-alpha-gamma-binding site-alpha
In contrast, Zhong et al’s was
beta-alpha-binding site-gamma-alpha-binding site-delta
What structure did Hansen et al study?
AChBP from Aplysia Californica; closely resembles L-AChBP (same protomer structure, key aromatic residues for binding were conserved)
How do A-AChBP and L-AChBP differ in their affinities for certain ligands?
A-AchBP has higher affinity for lobeline.
L-AchBP has higher affinity for Ach, nicotine, and epibatidine.
What structure did Hansen observe overlapped the most between muscle nAchRs and apo A-AchBPs?
Loop C (Double cys loop) in both structures overlap most closely.
What did Hansen et al. observe when comparing the apo A-AChBP with HEPES-bound A-AChBP?
The principal side of the C loop is closed more into the complementary side when bound with a HEPES buffer than when not bound.
What interactions does A-AChBP make with lobeline?
W147 (TrpB): there is hydrogen bonding between lobeline carbonyl oxygen hydrogens, and cation-pi bonding with lobeline.
The lobeline hydroxyl group hydrogen bonds with S149.
Other aromatic tyrosines (Y) point toward the lobeline amine.
What interactions does A-AChBP make with epibatidine?
W179 (TrpB): hydrogen bonding and cation-pi binding with the epibatidine bridge ring amine.
Other aromatic tyrosines (Y) point towards the epibatidine bridge ring amine.
How does A-AChBP bound with MLA compare to A-AChBP bound with alpha-conotoxin-IMI?
IMI is larger; the binding pocket expands to accomodate larger molecules. IMI binds higher up than MLA; this maintains hyperextension of the C-loop compared to the apo form.
When MLA is bound, the apo structure of A-AChBP is maintained (no hyperextension).
How does nicotine-bound L-AChBP compare to carbamylcholine? What is a key residue to consider.
Both nicotine and carbamycholine are oriented vertically in the binding pocket.
Both make close association with TrpB via cation-pi and hydrogen bonding.
D85: aspartic acid maintains the correct position of TrpB.
