Key residues in the nicotinic acetylcholine receptor β2 subunit contribute to α-conotoxin LvIA binding

Abstract

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr(59) and Phe(119) of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.

Keywords: Docking; Mutant α3β2 Subtype.; Receptor; Receptor Structure-Function; Receptor-interacting Protein (RIP); Toxin; α-Conotoxin LvIA; α3β2 nAChR; β subunit contribution.

FIGURE 1.

Reduced α-CTx LvIA synthetic intermediate (A) and oxidized, folded α-CTx LvIA (B) sequences and corresponding HPLC chromatograms (C and D). A, sequence of a synthetic intermediate of α-CTx LvIA with Cys² and Cys⁴ residues protected with S-acetamidomethyl (Acm), and Cys¹ and Cys³ residues with free -SH (mercapto) before initial cleavage. The first and third cysteine residues were initially protected with acid-labile groups (trityl), which were removed after cleavage from the resin. B, folded peptide sequences with disulfide connectivity 1–3, 2–4. *, C-terminal carboxamide. C, HPLC chromatograms of the synthetic intermediate shown in A. D, HPLC chromatograms of oxidized and folded α-CTX LvIA. Peptides were analyzed on a reversed-phase analytical Vydac C18 HPLC using a linear gradient of 0–40% Solvent B over 40 min, where Solvent A = 0.075% TFA and Solvent B = 0.05% TFA, 90% acetonitrile in H₂O. Absorbance was monitored at 214 nm. Flow rate was 0.75 ml/min. AU, absorbance units.

FIGURE 2.

Amino acid sequence alignment of rat β2 and β4 nAChR subunits, which have 68.4% sequence identity in their ligand-binding domains. The positions that were mutated in this study, i.e. positions 34, 59, 79, 111, and 119, are indicated with arrows. Rectangles indicate the agonist-binding domain loops D, E, and F (3). The subunit positions that were shown to contact LvIA according to a previous molecular modeling study (Luo et al. (25)) are underlined.

FIGURE 3.

α-CTx LvIA dose-response curves for wild-type and mutant α3β2 nAChRs. All seven mutant receptors exhibited similar sensitivity for ACh to wild-type α3β2 nAChR. Values are mean ± S.E. from a recording made using 5–9 separate oocytes. Results are summarized in Table 1.

FIGURE 4.

α-CTx LvIA differentially blocks wild-type α3β2 nAChR (A) and mutant receptors α3β2[V111I] (B), α3β2[F119Q] (C), and α3β2[T59K] (D). The nAChRs display different reversibility kinetics after block. C indicates control responses to ACh. Oocytes were exposed to 10 nm peptide for 5 min followed by repetitive application of ACh. α-CTx LvIA at 10 nm blocked ∼55% current of wild-type α3β2 nAChR with rapid reversibility (A), but did not block α3β2[V111I] nAChR (B). LvIA at 10 nm blocked ∼100% current of mutant receptors α3β2[F119Q] nAChR with slow reversibility (C) and α3β2[T59K] nAChR with slowest reversibility (D).

FIGURE 5.

Molecular modeling of the interaction between LvIA and α3β2 wild-type and mutant nAChR. A, binding of LvIA (white) into the rat wild-type α3β2-binding pocket, which comprises the α3 principal subunit (green) and the β2 complementary subunit (blue). The conformation of the side chains of the β2 positions that were mutated are displayed overlaid with those of the wild-type receptor. The side chains of the mutants are shown in different colors from those used for the wild-type structure. B, distance between the NZ atom of β2 Lys⁷⁹ and the CG atom of LvIA Asp¹¹ over a 2-ns molecular dynamics simulation. C, correlation between the differences of buried solvent-accessible surface area between wild-type and mutant complexes (Δ BASA) and the IC₅₀ for the α3β2 mutants.

FIGURE 6.

Molecular dynamics simulation of LvIA/α3β2 incorporating β2 subunit mutations T59K, V111I, or F119Q. A, overlay of the conformation of the binding sites after 10-ns simulations, with the α3 subunit in green, the β2 subunit in blue, and conotoxin LvIA in white. The side chains of LvIA as well as the mutated side chains Lys⁵⁹, Ile¹¹¹, and Gln¹¹⁹ are in stick representation. B, backbone root mean square deviation (RMSD) over the 10-ns simulations from the starting conformation of the β2 subunit binding site. This binding site is defined here as including the β1 (positions 32–40), β2 (positions 57–63), β5′ (positions 109–113), and β6 (positions 116–120) strands.