α-Conotoxin PeIA[S9H,V10A,E14N] potently and selectively blocks α6β2β3 versus α6β4 nicotinic acetylcholine receptors

Abstract

Nicotinic acetylcholine receptors (nAChRs) containing α6 and β2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of β2 and β4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind α6β2 nAChRs also potently bind the closely related α6β4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described α-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including α6/α3β2β3 and α6/α3β4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for α6/α3β2β3 nAChRs expressed in Xenopus oocytes (α6/α3 is a subunit chimera that contains the N-terminal ligand-binding domain of the α6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by α6/α3β2β3 and α6/α3β4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to α6/α3β2β3 nAChRs yielded a k(off) of 0.096 ± 0.001 min(-1) and a k(on) of 0.23 ± 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between α6β2 and α6β4 nAChRs.

Fig. 1.

Amino acid sequence comparison between α-Ctxs PeIA, MII, and PnIA. A, conserved cysteines are shown in bold; residues of PeIA that were selected for substitution with those of either MII or PnIA are shown underscored. B, concentration-response analysis of the activity of PeIA on Xenopus oocyte-expressed rat nAChRs. PeIA blocked I_ACh mediated by α3β2, α3β4, α6/α3β2β3, α6/α3β4, and α9α10 nAChRs with IC₅₀ (95% confidence interval) values of 9.73 (8.11–11.7) nM, 1.5 (1.3–1.7) μM, 11.1 (8.17–15.0) nM, 148 (124–176) nM, and 33.0 (28.0–33.9) nM, respectively. Error bars denote the S.E.M. of the data from three to five oocytes for each determination.

Fig. 2.

Concentration-response analysis of the singly substituted PeIA analogs PeIA[S9H] (A), PeIA[V10A] (B), and PeIA[E14N] (C) on Xenopus oocyte-expressed rat α3β2, α3β4, α6/α3β2β3, α6/α3β4, and α9α10 nAChRs. The error bars for the data denote the S.E.M. from four to eight oocytes for each determination. IC₅₀ values and confidence intervals are shown in Table 1.

Fig. 3.

Kinetic analysis of the activity of PeIA and single substituted analogs on Xenopus oocyte-expressed rat α3β2, α3β4, α6/α3β2β3, and α6/α3β4 nAChRs. The toxins were applied as described under Materials and Methods, and the data were fit to a single exponential equation. The error bars denote the S.E.M. of the data from three to nine oocytes for each determination. Note the different time scale used for PeIA[S9H]. See Table 2 for a summary of the values obtained.

Fig. 4.

Concentration-response analysis of the doubly substituted PeIA analogs PeIA[S9H,V10A] (A) and PeIA[S9H,E14N] (B) on Xenopus oocyte-expressed rat α3β2, α3β4, α6/α3β2β3, and α6/α3β4 nAChRs. The error bars for the data denote the S.E.M. from three to five oocytes for each determination. For a summary of the IC₅₀ values and confidence intervals see Table 3.

Fig. 5.

Concentration-response analysis of the activity of PeIA[S9H,V10A,E14N] on Xenopus oocyte-expressed nAChR subtypes. A, inhibition curves for rα6/α3β2β3, mα6/α3β2β3, hα6β2α4β2β3, rα6/α3β4, rα6β4, and mα6/α3β4 nAChRs. B, inhibition curves for rα3β2, rα3β4, rα4β2, rα4β4, rα7, and rα9β10 nAChRs. The error bars for the data denote the S.E.M. from four to five oocytes for each determination; r, rat; m, mouse; h, human. For a summary of the IC₅₀ values and confidence intervals see Table 4.

Fig. 6.

Comparison of PeIA[S9H,V10A,E14N] kinetics on rat α6/α3β2β3 versus α6/α3β4 nAChRs expressed in Xenopus oocytes. A, k_obs was determined for five concentrations of PeIA[S9H,V10A,E14N] from 100 pM to 5 nM by perfusing the oocytes with the toxin until a steady-state level of block was achieved. B, the data were fit to an exponential equation, and the observed rates are plotted as a function of the PeIA[S9H,V10A,E14N] concentration to obtain k_on. C, to obtain k_off, a high concentration of toxin was applied in a static bath for 5 min after which the perfusion was resumed, and the I_ACh was monitored for recovery (see Materials and Methods for the concentrations used). The same analysis was performed for α6/α3β4 nAChRs (D–F). The error bars in A to C denote the S.E.M. from four individual determinations and from five in D to F.

Fig. 7.

Block of α6/α3β2β3 and α6/α3β4 nAChRs by PeIA[S9H,V10A,E14N]. Oocytes expressing α6/α3β2β3 (A) and α6/α3β4 (B) nAChRs were continuously perfused with 5 nM PeIA[S9H,V10A, E14N] until a steady-state level of block was achieved; the percentage response was then quantified for comparison (C). The error bars in C denote the S.E.M. from four oocytes expressing the α6/α3β2β3 subtype and from six expressing the α6/α3β4 subtype. Significance was determined by a t test and compared with a theoretical response mean of 100%. ***, p < 0.001.

Fig. 8.

Comparison of the three-dimensional structures of PeIA[S9H,V10A,E14N], MII, and PnIA. A, α-Ctx PeIA[S9H,V10A,E14N] with residues substituted from MII shown in blue (His9) and (Asn14) and from PnIA shown in red (Ala10). Residues shown in white are nonhomologous with MII. B, α-Ctx MII with His9 and Asn14 shown in blue. C, α-Ctx PnIA with Ala10 shown in red. Images were generated using PyMOL as described under Materials and Methods.