Structure and function of μ-conotoxins, peptide-based sodium channel blockers with analgesic activity

Abstract

μ-Conotoxins block voltage-gated sodium channels (VGSCs) and compete with tetrodotoxin for binding to the sodium conductance pore. Early efforts identified µ-conotoxins that preferentially blocked the skeletal muscle subtype (NaV1.4). However, the last decade witnessed a significant increase in the number of µ-conotoxins and the range of VGSC subtypes inhibited (NaV1.2, NaV1.3 or NaV1.7). Twenty µ-conotoxin sequences have been identified to date and structure-activity relationship studies of several of these identified key residues responsible for interactions with VGSC subtypes. Efforts to engineer-in subtype specificity are driven by in vivo analgesic and neuromuscular blocking activities. This review summarizes structural and pharmacological studies of µ-conotoxins, which show promise for development of selective blockers of NaV1.2, and perhaps also NaV1.1,1.3 or 1.7.

Figure 1. Voltage-gated sodium channels structure

(A) Crystal structure of the bacterial sodium channel Na_VAb (PDB code 4EKW). Structure illustrates the four homologous domains of the channel (DI-DIV) arranged around the highly selective pore region through which Na⁺ permeates. (B) Individual domain comprising six membrane-spanning subunits (S1–S6) with the site of action (P-loop site 1) for μ-conotoxins discussed throughout this review [21]. (C) Cartoon of the VGSC α- and β-subunits. Selectivity filter is formed by the looped regions between S5 and S6 (i.e., P-loop). Approximate locations of neurotoxin-binding Sites 1-5 are shown on the α-subunit. Site 1, the location of μ-conotoxin binding, is emphasized. β-subunit crystal structure from Gilchrist et al. (PDB code 4MZ2) [26]. VGSC: Voltage-gated sodium channels.

Figure 2. Examples of small molecule inhibitors of voltage-gated sodium channels

^†Indicates clinically used voltage-gated sodium channels. Data taken from [27].

Figure 3. The μ-conotoxins as an emerging class of sodium channel blocking peptides

Timeline illustrates the relative discoveries and/or characterization of the members of this family. Data taken from [53].

Figure 4. Summary of identified μ-conotoxins from m4 and m5 branches of the M-superfamily

ER signal and propeptide sequences for μ-GIIIA and μ-SIIIA are illustrated as examples. ‘O’ denotes hydroxyproline; ‘Z’ denotes pyroglutamic acid. Arg13 in μ-GIIIA, or the residue in the equivalent position, is underlined to illustrate the sequence differences among conotoxins that preferentially block muscle versus neuronal subtypes. ER: Endoplasmic reticulum Adapted with permission from [16,59].

Figure 5. Selectivity profiles of μ-conotoxins against Na_V1-subtypes

Data were obtained from reported IC₅₀values; *represents data obtained from pK_d values [53]. Pain-relevant subtypes are highlighted in red. Broken bars represent values greater than 100 μM. For color images please see online http://www.future-science.com/doi/full/10.4155/FMC.14.107

Figure 6. Structure–activity characterization of μ-conotoxins

Potencies of characterized μ-conotoxins against skeletal (Na_V1.4) and neuronal (Na_V1.2) subtypes. Solution structures showing the folded peptide structures with disulfide connectivity. PDB ID# 1TCK (μ-GIIIA), 1R9I (μ-PIIIA), 1Q2J (μ-SmIIIA), 2LXG (μ-KIIIA) and 2LO9 (μ-BuIIIB). BMRB Entry# 20024 (μ-TIIIA) and 20023 (μ-SIIIA). Results of individual amino acid replacements on Na_V1-subtype blockade. (↑) indicates mutations that improve potency against Na_V1-subtypes, (↓) denotes decreased potency and (°) represents no change in potency. (desZ1) indicates deletion of pyroglutamic acid in position 1 of μ-SIIIA.

Figure 6. Structure–activity characterization of μ-conotoxins

Potencies of characterized μ-conotoxins against skeletal (Na_V1.4) and neuronal (Na_V1.2) subtypes. Solution structures showing the folded peptide structures with disulfide connectivity. PDB ID# 1TCK (μ-GIIIA), 1R9I (μ-PIIIA), 1Q2J (μ-SmIIIA), 2LXG (μ-KIIIA) and 2LO9 (μ-BuIIIB). BMRB Entry# 20024 (μ-TIIIA) and 20023 (μ-SIIIA). Results of individual amino acid replacements on Na_V1-subtype blockade. (↑) indicates mutations that improve potency against Na_V1-subtypes, (↓) denotes decreased potency and (°) represents no change in potency. (desZ1) indicates deletion of pyroglutamic acid in position 1 of μ-SIIIA.

Figure 7. Description of the ddSec strategy used to facilitate structure–activity relationship studies of μ-BuIIIB

(A) Cartoon depiction of μ-BuIIIB showing disulfide connectivity between cysteine thiols. (B) Cartoon of ddSecBuIIIB scaffold with disulfide depletion by removal of the Cys6–Cys23 bridge and diselenide bridge formation by selenocysteine replacement of Cys5 and Cys17. (Panels C and D) Upper and lower chromatograms show linear and folded elution patterns of μ-BuIIIB (C) and ddSecBuIIIB (D), respectively. *Indicates the properly folded isoform. Positional scanning of all noncysteine residues was subsequently performed using the ddSecBuIIIB scaffold. Reproduced with permission from [85] © 2014 FEBS.

Figure 8. Conotoxins at various stages of preclinical and clinical development

n.a.: Not available Data taken from[9,132].