Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3β2β3 and α3β2 nicotinic acetylcholine receptors

Abstract

The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for α3β2 but not for α6/α3β2β3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3β2β3 than α3β2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu(152) of the α6 subunit and Lys(152) of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6β2* nAChRs.

Keywords: Electrophysiology; Neurotoxin; Neurotransmitter Receptors; Nicotinic Acetylcholine Receptors; Oocyte; alpha-Conotoxin; alpha3beta2; alpha6beta2.

FIGURE 1.

Sequence alignment of α-Ctxs PeIA, MII, TxIB, OmIA, and PnIA. Amino acids highlighted in boldface type are non-conserved among the five species.

FIGURE 2.

Effect of Ala or Hyp substitutions of non-cysteine amino acids on the potency of α-Ctx PeIA for rat α6/α3β2β3 and α3β2 nAChRs. Xenopus oocytes expressing α6/α3β2β3 or α3β2 nAChRs were subjected to TEVC as described under “Experimental Procedures,” and the IC₅₀ values for inhibition of the I_ACh by each PeIA analog were determined. A and B, concentration-response analysis for inhibition of α3β2 and α6/α3β2β3 nAChRs by PeIA analogs with Ala or Hyp substitutions in the first Cys loop. C and D, concentration-response analysis for inhibition of α3β2 and α6/α3β2β3 nAChRs by PeIA analogs with Ala or Hyp substitutions in the second Cys loop. The IC₅₀ values are summarized in Table 1. Error bars, S.E. from 4–5 oocytes for each experimental determination.

FIGURE 3.

Effect of non-cysteine amino acid substitutions on the potency of α-Ctx PeIA for rat α6/α3β2β3 and α3β2 nAChRs. Xenopus oocytes expressing α6/α3β2β3 or α3β2 nAChRs were subjected to TEVC as described under “Experimental Procedures,” and the IC₅₀ values for inhibition of the I_ACh by each PeIA analog were determined. A and B, concentration-response analysis for inhibition of α3β2 and α6/α3β2β3 nAChRs by PeIA analogs with substitutions from MII. C and D, positional scanning of residues 9, 10, and 11 of PeIA with positively charged amino acids and the concentration-response curves for inhibition of α3β2 and α6/α3β2β3 nAChRs by the resulting analogs. The IC₅₀ values are summarized in Table 2. Error bars, S.E. from 4–5 oocytes for each experimental determination.

FIGURE 4.

Effect of combined substitutions of non-cysteine amino acids on the potency of α-Ctx PeIA for rat α6/α3β2β3 and α3β2 nAChRs. Xenopus oocytes expressing α6/α3β2β3 or α3β2 nAChRs were subjected to TEVC as described under “Experimental Procedures,” and the IC₅₀ values for inhibition of the I_ACh by each PeIA analog were determined. A and B, concentration-response analysis for inhibition of α3β2 and α6/α3β2β3 nAChRs by PeIA(S9H,V10A,N11R), PeIA(A7V,S9H,V10A,N11R), and PeIA(S9H,V10A,N11R,E14A). The IC₅₀ values are summarized in Table 3. Error bars, S.E. from 4–5 oocytes for each experimental determination.

FIGURE 5.

Potency and selectivity profile of PeIA(A7V,S9H,V10A,N11R,E14A) for several rat nAChR subtypes. Xenopus oocytes expressing different nAChRs were subjected to TEVC as described under “Experimental Procedures,” and the IC₅₀ values for inhibition of the I_ACh by PeIA(A7V,S9H,V10A,N11R,E14A) were determined. A, concentration-response analysis for inhibition of α6/α3β2β3 and α3β2 nAChRs. B, concentration-response analysis for inhibition of α3β4, α4β2, α4β4, α6β4, and α7 nAChRs. In B, the 10 μm data points for α3β4, α4β2, α4β4, and α7 nAChRs are shown staggered to avoid overlap. The IC₅₀ values are summarized in Table 4. Error bars, S.E. from 4–5 oocytes for each experimental determination.

FIGURE 6.

The ligand binding kinetics of block and unblock of α6/α3β2β3 and α3β2 nAChRs by PeIA(A7V,S9H,V10A,N11R,E14A) at three progressively higher toxin concentrations. A, in the presence of 100 nm PeIA(A7V,S9H,V10A,N11R,E14A), the average response to 100 μm ACh was 2.7 ± 0.3% (n = 3) of control responses for α6/α3β2β3 receptors. B, for α3β2 receptors, the average response to 100 μm ACh in the presence of 100 nm and 1 μm PeIA(A7V,S9H,V10A,N11R,E14A) was 98.3 ± 2.2% (n = 4) and 87.0 ± 3.5% (n = 4), respectively. Values are ± S.E. C, control.

FIGURE 7.

Sequence alignment of rat nAChR subunits. A, sequence alignment of the N-terminal ligand binding domains of α6 and α3. A pairwise comparison of the sequences of the two subunits identified 137 (67%) identities and 32 (15%) similarities at the amino acid level. The arrows indicate the three amino acids of the α6 and α3 ligand binding domains that were examined in this study using mutagenesis. B, the first 210 amino acid pairs of the β2 and β3 subunits contained 81 (38%) identities and 22 (21%) similarities. The asterisks in B identify residues of the β2 subunit that have been shown previously to be important for α-Ctx binding (19, 47).

FIGURE 8.

The sensitivity of α6/α3β2β3 and α3β2 nAChRs to inhibition by PeIA(N11R) and PeIA(A7V,S9H,V10A,N11R,E14A) is determined by three residues in the α6/α3 and α3 subunits. Xenopus oocytes expressing α6/α3β2β3, α3β2, and mutants of these receptors were subjected to TEVC as described under “Experimental Procedures,” and the inhibition of the I_ACh by PeIA(N11R) and PeIA(A7V,S9H,V10A,N11R,E14A) was determined. The IC₅₀ values are summarized in Table 5. Error bars, S.E. from 4–5 oocytes for each experimental determination.

FIGURE 9.

Homology binding model of PeIA(A7V,S9H,V10A,N11R,E14A) to rat α3β2 and α6β2 nAChRs. A, a 20 Å radius view of the ACh binding pocket of the A. californica AChBP complexed with α-Ctx PnIA(A10L,D14K) The principal subunit is shown in green, and the complementary subunit is shown in cyan. Residues 147–153 of the B-loop and 185–195 of the C-loop of the principal binding subunit were mutated to the homologous residues found in the rat α3 subunit. Residues of the C-loop shown in yellow are Cys¹⁸⁹ and Cys¹⁹⁰, and the residues shown in blue are Glu¹⁸⁴ and Gln¹⁹⁵ of the C-loop, and Lys¹⁵² of the B-loop, shown as a stick model. Residues of PnIA(A10L,D14K) were mutated to those of PeIA(A7V,S9H,V10A,N11R,E14A) and shown in red with Arg¹¹ shown as a stick model. Note the close proximity of Lys¹⁵² of the α3 subunit and Arg¹¹ of the α-Ctx, suggesting a possible interaction between the two. B, residues 147–153 of the B-loop and 185–195 of the C-loop of the AChBP principal binding subunit were mutated to the homologous residues found in the rat α6 subunit. Residues in blue are Asp¹⁸⁴ and Thr¹⁹⁵ of the C-loop and Glu¹⁵² of the B-loop. Mutagenesis of the AChBP and α-Ctx PnIA(A10L,D14K) as well as image rendering were performed as described under “Experimental Procedures.”

FIGURE 10.

The sensitivity of rat α6β4 to inhibition by PeIA, PeIA(S9A), and PeIA(S9R). Xenopus oocytes expressing α6β4 nAChRs were subjected to TEVC as described under “Experimental Procedures,” and the IC₅₀ values for inhibition by PeIA and two analogs were determined. PeIA, PeIA(S9A), and PeIA(S9R) inhibited α6β4 with IC₅₀ values of 262 (201–340) nm, 1.18 (0.947–1.147) μm, and 4.46 (3.30–6.02) μm, respectively. Values in parenthesis indicate the 95% confidence interval; error bars, S.E. from 4–5 oocytes for each experimental determination.