Midterm 2 Content - nAChRs and AChBP

Question 1

What is the structure of a nAchR agonist typically like? What are nAchR agonists/partial agonists/antagonists?

Answer 1

Quaternary ammoniums (N-R4+) with a positive charge.

Full agonists: nicotine, Ach, Epibatidine, Carbacholamine
Partial agonists: lobeline
Antagonists: MLA (Methylylcaconitine) and Alpha-conotoxin-IMI.

Question 2

What are the different types of nAchRs and what subunits can they consist of?

Answer 2

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.

Question 3

What are the 3 domains of an nAchR?

Answer 3

Extracellular ligand-binding domain
Transmembrane domain
Cytoplasmic/Intracellular domain

Question 4

What is the membrane topology of a nAChR like?

Answer 4

See notes for drawing
n = 5
4 transmembrane regions
M2 is pore-lining

Question 5

What are the binding sites of muscle nAchRs like?

Answer 5

Between alpha-gamma/epsilon inferface and alpha-delta interface
Binding sites have A, B, C, D, E, and F loops.

Question 6

What are the binding sites of neuronal nAchRs like?

Answer 6

Homopentamers: 5 binding sites, at every subunit interface.
Heteropentamers: 2 binding sites, each between an alpha and beta subunit.

Question 7

What kind of receptor did Zhong et al. look at?

Answer 7

Muscle nAChR

Question 8

How did Zhong et al make unnatural amino acids?

Answer 8

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.

Question 9

How do different side chains have an effect on cation-pi binding?

Answer 9

The addition of more electronegative residues pulls electrons away from the aromatic “electron cloud” and the binding energy of the cation-pi bond decreases.

Question 10

What is EC50?

Answer 10

Effective concentration 50%: the concentration of agonist at which 50% of the maximal response is received.

Question 11

What is the relationship between EC50 and cation-pi bonding between Ach and nAChRs? Is this changed with nicotine?

Answer 11

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.

Question 12

What residue did Zhong et al discover made the only cation-pi interaction with Ach?

Answer 12

Trp149 (W149) on the B loop of the binding pocket (TrpB)

Question 13

What did Zhong et al. observe when they tethered a quaternary amine to TrpB?

Answer 13

This resulted in a constitutively active nicotine current.

Question 14

What receptor did Xiu et al. study?

Answer 14

Alpha4beta2 neuronal nicotinic receptors.

Question 15

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?

Answer 15

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.

Question 16

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?

Answer 16

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.

Question 17

What is AchBP? What organism is it obtained from and what does it resemble?

Answer 17

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.

Question 18

Where is AchBP found naturally in the body?

Answer 18

Released in an Ach-dependent manner into the synaptic cleft by some glia to modulate synaptic transmission?

Question 19

What is alpha bungarotoxin?

Answer 19

An AchBP antagonist; neurotoxin found in snakes.

Question 20

What did Brejc study?

Answer 20

The AchBP of Lymnaea stagnalis

Question 21

What is the structure of AchBP like? What does it resemble?

Answer 21

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”

Question 22

What does an AChBP protomer consist of?

Answer 22

N-terminal alpha helix
2 310 helices
10 beta strands (a beta sandwich)

Question 23

What is a Cys loop made of?

Answer 23

A loop consisting of highly conserved amino acids between two cysteine residues, which form a disulphide bond.

Question 24

What is the main immunogenic region (MIR)?

Answer 24

A region of muscle nAChRs that acts as an epitope in Myasthenia Gravis.

Question 25

What is the double Cys loop?

Answer 25

Another name for the C loop of nAchRs/AchBP; “sticks out” from the AchBP protomer.

Question 26

What is Myasthenia Gravis? What are symptoms and treatments?

Answer 26

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.

Question 27

What is the structure of the AChBP ligand-binding site like?

Answer 27

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.

Question 28

What is HEPES?

Answer 28

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.

Question 29

How did Brejc et al. correct Zhong et al’s model of the muscle nAChR stoichiometry?

Answer 29

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

Question 30

What structure did Hansen et al study?

Answer 30

AChBP from Aplysia Californica; closely resembles L-AChBP (same protomer structure, key aromatic residues for binding were conserved)

Question 31

How do A-AChBP and L-AChBP differ in their affinities for certain ligands?

Answer 31

A-AchBP has higher affinity for lobeline.

L-AchBP has higher affinity for Ach, nicotine, and epibatidine.

Question 32

What structure did Hansen observe overlapped the most between muscle nAchRs and apo A-AchBPs?

Answer 32

Loop C (Double cys loop) in both structures overlap most closely.

Question 33

What did Hansen et al. observe when comparing the apo A-AChBP with HEPES-bound A-AChBP?

Answer 33

The principal side of the C loop is closed more into the complementary side when bound with a HEPES buffer than when not bound.

Question 34

What interactions does A-AChBP make with lobeline?

Answer 34

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.

Question 35

What interactions does A-AChBP make with epibatidine?

Answer 35

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.

Question 36

How does A-AChBP bound with MLA compare to A-AChBP bound with alpha-conotoxin-IMI?

Answer 36

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).

Question 37

How does nicotine-bound L-AChBP compare to carbamylcholine? What is a key residue to consider.

Answer 37

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.