Structure and activity of alpha-conotoxin PeIA at nicotinic acetylcholine receptor subtypes and GABA(B) receptor-coupled N-type calcium channels

Abstract

α-Conotoxins are peptides from cone snails that target the nicotinic acetylcholine receptor (nAChR). RgIA and Vc1.1 have analgesic activity in animal pain models. Both peptides target the α9α10 nAChR and inhibit N-type calcium channels via GABA(B) receptor activation, but the mechanism of action of analgesic activity is unknown. PeIA has previously been shown to inhibit the α9α10 and α3β2 nAChRs. In this study, we have determined the structure of PeIA and shown that it is also a potent inhibitor of N-type calcium channels via GABA(B) receptor activation. The characteristic α-conotoxin fold is present in PeIA, but it has a different distribution of surface-exposed hydrophobic and charged residues compared with Vc1.1. Thus, the surface residue distribution, rather than the overall fold, appears to be responsible for the 50-fold increase in selectivity at the α3β2 nAChR by PeIA relative to Vc1.1. In contrast to their difference in potency at the nAChR, the equipotent activity of PeIA and Vc1.1 at the GABA(B) receptor suggests that the GABA(B) receptor is more tolerant to changes in surface residues than is the nAChR. The conserved Asp-Pro-Arg motif of Vc1.1 and RgIA, which is crucial for potency at the α9α10 nAChR, is not required for activity at GABA(B) receptor/N-type calcium channels because PeIA has a His-Pro-Ala motif in the equivalent position. This study shows that different structure-activity relationships are associated with the targeting of the GABA(B) receptor versus nAChRs. Furthermore, there is probably a much more diverse range of conotoxins that target the GABA(B) receptor than currently realized.

FIGURE 1.

Sequences of α-conotoxins that target the α9α10 nAChR. The cysteine residues are connected in the native disulfide connectivity as indicated. The asterisk represents an amidated C terminus.

FIGURE 2.

NMR chemical shifts and structure of PeIA. A, the secondary shifts of PeIA (red) and Vc1.1 (blue) were calculated by subtracting random-coil α-H shifts (33) from the experimental shifts. The negative secondary shifts for residues 6–11 are indicative of helical structure. The similarities in secondary shifts between the two peptides indicate that the three-dimensional structures are similar. B, superposition of the 20 lowest energy structures of PeIA (red) and Vc1.1 (blue). Despite the majority of secondary shifts being negative, only residues 6–10 are formally recognized as an α-helix. Similarly, in the structures calculated previously for Vc1.1, only residues 6–12 are recognized as an α-helix despite the negative secondary shifts at the N and C termini (30). It appears that the turn regions at the N and C termini of PeIA and Vc1.1 result in the negative secondary shifts present in these regions. C, surface representations of PeIA (left) and Vc1.1 (right). Hydrophobic residues are shown in green, cysteines in yellow, polar in cyan, positively charged in blue, and negatively charged residues in red.

FIGURE 3.

Effect of α-conotoxin PeIA on nAChR subtypes expressed in Xenopus oocytes and GABA_B-mediated inhibition of N-type Ca²⁺ channels in rat DRG neurons. A, representative superimposed currents evoked by ACh in α9α10, α3β2, and α7 nAChRs expressed in oocytes in the absence (Control) and presence of 100 nm and 1 μm PeIA. Rat α9α10 and α3β2 nAChRs were activated by 30 μm and 100 μm ACh, respectively, whereas human α7 nAChRs were activated by 200 μm ACh. B, concentration-response relationships obtained for the inhibition of ACh-evoked current amplitudes following 5 min of incubation of α9α10 (●) and α3β2 (○) nAChRs with PeIA giving IC₅₀ values of 54.9 and 97.5 nm, respectively. No significant inhibition was observed with concentrations up to 1 μm PeIA at α4β2 (▾), human α7 (△) and muscle αβγδ (■) nAChRs. Concentration-response data (means ± S.E., n = 4–7 for each data point) were fitted using the logistic equation (see “Experimental Procedures”).

FIGURE 4.

Effect of α-conotoxin PeIA on GABA_B-mediated inhibition of N-type Ca²⁺ channels in rat DRG neurons. A, superimposed depolarization-activated whole-cell Ba²⁺ currents elicited by voltage steps from a holding potential of −80 to −10 mV in the absence (Control) and presence of 10 nm PeIA. B, concentration-response relationship obtained for inhibition of HVA Ca²⁺ channel currents in DRG neurons by PeIA (n = 4–13 cells for each data point) giving an IC₅₀ of 1.1 nm. The effect of PeIA was antagonized in the presence of 100 and 300 nm CGP55845 (n = 3). Data points represent means ± S.E. of normalized peak current amplitude. C, representative time course of inhibition of peak Ba²⁺ current amplitude in the presence of the selective N-type Ca²⁺ channel inhibitor ω-conotoxin CVID, followed by application of PeIA (100 nm). pF, picofarads. D, bar graph of the relative inhibition of HVA Ca²⁺ channel currents by 100 nm PeIA alone, in the presence of 1 μm CGP55845 alone, and after application of 100 nm PeIA in the presence of CGP55845. Numbers in parentheses reflect the number of cells. PeIA significantly reduced the HVA Ca²⁺ channel currents compared with CGP55845 and CGP55845 + PeIA in an unpaired t test (p < 0.0001). Cells treated with CGP55845 alone and PeIA + CGP55845 were not significantly different in a paired t test (p = 0.312).