A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors

Abstract

Background and purpose: The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant.

Experimental approach: µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data.

Key results: Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V)1.4 (IC(50) = 1.3 nM) and Na(V)1.2 channels in a long-lasting manner. Cardiac Na(V)1.5 and DRG-specific Na(V)1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3β2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs.

Conclusion and implications: µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

Figure 1

Isolation and characterization of µ-CnIIIC. (A) Reverse-phase HPLC chromatogram (monitored by absorbance at 214 nm) of the venom duct extract from Conus consors. The peak eluting at ∼28 min contained µ-CnIIIC that was purified to homogeneity at an analytical scale and showed a single monoisotopic molecular mass (inset). The peptide was then subjected to reduction and mass spectrometry analysis. (B) ESI-MS/MS spectrum of the the m/z 596 undergoing collision-induced dissociation. The good fragmentation coverage allowed complete amino acid sequence assignment with y and b ions.

Figure 2

Effects of different µ-conotoxins on isometric twitch tension elicited by direct electrical stimulation of isolated mouse EDL muscles. (A) Effects of µ-conotoxins on skeletal muscle contraction. Contractions evoked by direct muscle stimulation and recorded in the absence and presence of 0.1 µM µ-conotoxins. Scale bars: 0.2 g (vertical) and 0.1 s (horizontal). (B) Concentration–response curves of peptide effects on directly elicited muscle contraction. For each µ-conopeptide concentration, the maximal twitch peak amplitude is expressed relative to its control value as the mean ± SEM of three to six experiments. The theoretical curves were calculated from typical sigmoidal nonlinear regression through data points (correlation coefficient r²≥ 0.948). The µ-conopeptide concentration that inhibited 50% of the twitch tension (IC₅₀) is indicated for each peptide studied.

Figure 3

Effect of µ-CnIIIC on the GAP recorded on isolated mouse sciatic and pike olfactory nerves. (A–B) Traces of GAP recorded in response to 0.05 ms (mouse) and 8 ms (pike) and 0.1–15 V stimulations, under control conditions and after treatment with µ-CnIIIC at the indicated concentrations. (C–D) GAP amplitude in response to different intensities of 0.05 and 8 ms stimulations and to different concentrations of µ-CnIIIC. (E–F) GAP amplitude in the presence of various concentrations of µ-CnIIIC, and expressed relative to control values as mean ± SEM of three to six mouse sciatic and three to four pike olfactory nerves. The curves were calculated from typical sigmoidal nonlinear regression through data points (r²≥ 0.984). The µ-CnIIIC concentrations required to block 50% of the GAP amplitude (IC₅₀) was 1.53 µM (mouse) and 0.15 µM (pike).

Figure 4

Effect of µ-CnIIIC on voltage-gated sodium channels. (A) Sodium currents at −20 mV recorded from HEK 293 cells expressing rat Na_V1.4 channels, under control conditions and in the presence of 1 µM µ-CnIIIC (red). Holding potential was −120 mV, leak was corrected with a p/6 method. The panel on the right shows currents in the presence of toxin scaled and superimposed on the control current, indicating a lack of kinetic changes. (B) Na_V1.4 peak current inhibition by µ-CnIIIC and wash-out with a conotoxin-free medium shown as a function of time. The continuous red curve is a single exponential; filled circles indicate data points corresponding to traces shown in (A). (C) The remaining current after toxin equilibration (left) as a function of µ-CnIIIC concentration and the inverse of the time constant characterizing the onset of block (right) as a function of µ-CnIIIC concentration. The number of experiments is indicated in parentheses. The continuous lines are global data fits assuming a first-order reaction yielding an apparent IC₅₀ value of 1.3 ± 0.4 nM and a negligible off-rate. Error bars indicate SEM. (D) Current traces as in A for the indicated channel types before (black) and after application of µ-CnIIIIC at the indicated concentration (red). Traces for rat Na_V1.8 channels were recorded from Neuro-2A cells at 10 mV in the presence of 1 µM TTX.

Figure 5

Effect of µ-CnIIIC on nAChRs expressed in Xenopus oocytes. (A1-A3) presents the inhibition of acetylcholine-induced currents on human α7 (A1), α4β2 (A2) and α3β2 (A3) nAChRs. All recordings were carried out at −100 mV and the first three traces are controls followed by concentration-dependent response to 2 min exposure to different concentrations of µ-CnIIIC, ranging from 1 nM to 10 µM. Each experiment was terminated by wash-out steps for a time period of 8 min. Complete wash-out was observed for α7 and α4β2, while only partial recovery occurred for α3β2. (B1–3) Inhibition curves of the fitted data. IC₅₀, Hill coefficient (n_H) and experimental numbers are presented under the fitted curve for α7 (B1), α4β2 (B2) and α3β2 (B3). Error bars indicate SEM.

Figure 6

Structure of µ-CnIIIC. (A) Backbone superimposition over residues 3–22 for the 20 lowest energy structures (ribbon). (B) Lowest energy structure with the disulfide bridge shown in yellow. (C) Backbone superimposition of N-terminal region (residues 3–11) of µ-CnIIIC (blue) with N-terminal region (residues 4–12) of µ-TIIIA (red); Pro⁷ and Hyp⁸ of µ-CnIIIC and µ-TIIIA respectively are highlighted (sticks). (D) Backbone superimposition of C-terminal region (residues 12–22) of µ-CnIIIC (blue) with C-terminal region (residues 10–20) of µ-SIIIA (orange); Lys¹³ and Lys¹¹ of µ-CnIIIC and µ-SIIIA respectively are highlighted (sticks). Structural data were used from biological magnetic resonance bank (BMRB) accession codes 20024 (TIIIA) and 20025 (SIIIA). µ-CnIIIC structural data can be accessed with protein databank (PDB) code 2YEN and BMRB accession code 17581.

Figure 7

Structure comparison of µ-CnIIIC with α-conotoxins. (A–C) Backbone superimposition over Cα atoms of µ-CnIIIC (blue) with α-AuIB (grey), α-PnIA (magenta) and α-Vc1.1 (green). Disulfide bonds are shown in yellow for µ-CnIIIC and orange for the α-conotoxins. Protein databank (PDB) codes are 1DG2 (AuIB), 1PEN (PnIA) and 2H8S (Vc1.1).