A Conus regularis conotoxin with a novel eight-cysteine framework inhibits CaV2.2 channels and displays an anti-nociceptive activity

Abstract

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits CaV2.2 channel current in a dose-dependent manner with an EC50 of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for CaV2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.

Figure 1

Purification of RsXXIVA by reversed-phase high-performance liquid chromatography (RP-HPLC). All purification protocols were conducted at room temperature; the elution was monitored for absorbance at 230 nm. Panel A: fractionation of the venom extract from C. regularis by an analytical C₁₈ column eluted with a linear gradient from 0% to 60% of buffer B, over 60 min at 1 mL/min. Panel B: the peak indicated by the arrow in panel A was further purified on a micro-bore C₁₈ column using a linear gradient from 10% to 30% of buffer B, over 60 min at 200 μL/min. Panel C: the amino acid sequence of RsXXIVA was determined by mass spectrometry, and the amino terminal sequence was confirmed by automated Edman degradation (underlined amino acids).

Figure 2

Sequence comparison. Comparison of the RsXXIVA and MVIIA conotoxins found in C. regularis and C. magus, respectively. Identical residues (*), conserved substitutions (:) and semi-conserved substitutions (.) among these conotoxins are shown along the bottom.

Figure 3

Dose-response relationship for calcium current inhibition by RsXXIVA from C. regularis venom. (A) Relative superimposed calcium current traces under control conditions and under RsXXIVA application at different concentrations are shown at the top of every current trace. Dotted lines correspond to the zero current and the maximum current. (B) Symbols represent the average percentage of current inhibition at each toxin concentration, plotted on a semi-logarithmic scale. Data were fitted to a sigmoid function (solid line), with the following equation: y = [(A₁ − A₂)/{1 + ([Tx]/[Tx]₀)ⁿ}] + A₂. The midpoint value, [Tx]₀, was 2.8 μM, which corresponds to EC₅₀, and the Hill coefficient calculated from this set of data was n = 0.94.

Figure 4

Time course of Ca_V2.2 current inhibition by application of 3 μM of RsXXIVA in rat superior cervical ganglion (SCG) neurons. (A) Symbols are mean calcium currents of the test pulse before (1), during (2) and after (3) toxin application. The test pulse was delivered every 4 s, and at the end of the experiment, 100 μM CdCl₂ was applied (4). The inset is representative of calcium currents for each condition. (B) The summary of inhibition and recovery (wash) of calcium current under toxin RsXXIVA application. * Represents p < 0.05.

Figure 5

Effect of RsXXIVA (0.85 mg/kg intraperitoneal (IP)) in the hot-plate test. Adult male imprinting control region (ICR) mice where IP injected with 200 μL of either toxin or Nalbufin (4 mg/kg) as the positive control and PBS 1× as the negative control. Data are expressed as the mean ± SEM (n = 3). (A) The biological effect of RsXXIVA at 30 min post-injection. RsXXIVA shows (with 95% CI = 28.29 ± 3.35) an analgesic effect in acute thermal pain at 30 min post-injection in reference to the control group. (B) The biological effect of RsXXIVA 45 min post-injection. RsXXIVA shows an analgesic effect in acute thermal pain at 45 min post-injection (with 95% CI = 28.10 ± 3.71). Data were compare by one-way ANOVA, followed by Dunnett’s multiple comparison test * represents p < 0.05, considered as significant.

Figure 6

Effect of RsXXIVA (0.85 mg/kg IP) in formalin test. Adult male ICR mice where subcutaneously (SC) injected with 2.5% formalin (20 μL) 15 min before IP administration of 200 μL of either toxin or control Ketorolac (10 mg/kg) and Tramadol (5 mg/kg) as the positive control and PBS 1× as the negative control. Data are expressed as the mean ± SEM (n = 3). (A) The biological effect during phase 1 (0–5 min). RsXXIVA reduced the licking time compared with the control group (with 95% CI = 24.33 ± 14.69), reflecting activity in response to acute pain. (B) The biological effect during phase 2 (11–45 min). RsXXIVA appeared to have an anti-nociceptive effect compared with the control group (with 95% CI = 50.05 ± 13.33), which reflects chronic pain. Data were analyzed with one-way ANOVA, followed by Dunnett’s multiple comparison test. * Represents p < 0.05, considered as significant.