Midterm 2 Content - nAChRs and AChBP
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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.