Beta2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor.
The structures of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha3beta2 model to identify beta2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha3beta2 nAChR by two-electrode voltage clamp analysis. Although a beta2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta2-F117A, beta2-V109A, and beta2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta2-F117A mutant was combined with the alpha4 instead of the alpha3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta2-F117A, beta2-V109A, and beta2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha3beta2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.
Dutertre
Sébastien
S
Institute for Molecular Bioscience, The University of Queensland, Queensland 4072, Australia.
Nicke
Annette
A
Lewis
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020916:Conotoxins / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D020128:Inhibitory Concentration 50; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014981:Xenopus; D014982:Xenopus laevis
D020916:Conotoxins; D018076:DNA, Complementary; D011978:Receptors, Nicotinic; D007930:Leucine