Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception
Data files
Feb 24, 2026 version files 55.53 KB
Abstract
Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a neurotensin analogue, has been demonstrated to be safe and potentially analgesic in humans. However, the mechanism of action for CGX analgesia is unknown. We hypothesized that spinal application of CGX produces antinociception through activation of the presynaptic neurotensin receptor (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception in rodent models of inflammatory and neuropathic pain. Intrathecal administration of CGX, dose dependently, inhibited thermal and mechanical hypersensitivities in rodents of both sexes. Pharmacological and clustered regularly interspaced short palindromic repeats/Cas9 editing of NTSR2 reversed CGX-induced antinociception without affecting morphine analgesia. Electrophysiological and gene editing approaches demonstrated that CGX inhibition was dependent on the R-type voltagegated calcium channel (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic fractionation and slice electrophysiology recordings confirmed a predominantly presynaptic effect. Together, these data reveal a nonopioid pathway engaged by a human-tested drug to produce antinociception.
Dataset DOI: 10.5061/dryad.fxpnvx161
Description of the data and file structure
There are 6 figures in this paper and the data in the excel sheet is organized by figure.
figure 1:
in the excel sheet under figure 1 is the raw numerical outputs for the von frey animal behavior. and area under the curve. the data is listed under the corresponding panel
Panel B and C:
Row Labels:
Animals: numerically represented, but placed by male and female groups each with experimental treatment variables ( Saline, CGX 0.02nmol, CGX 0.1nmol, CGX 0.5nmol)
BL = Baseline measurement (before surgery).
PostSx = 24 hours post-surgery, immediately before treatment injection. 0.5, 1, 2, 4, 5 = Hours after treatment injection.
And for the units:
Units:
All values in Panel B (von Frey test) are reported as [e.g., withdrawal threshold in grams].
All values in Panel C (Hargreaves test) are reported as [e.g., paw withdrawal latency in seconds].
Panel D: Area under the curve analysis for mechanical withdrawal thresholds is presented in panel (B)
Panel E: Area under the curve analysis for thermal withdrawal latencies id presented in panel (C).
The AUC was calculated from the time of injection (time 0, which corresponds to the "PostSx" baseline) through the 5-hour time point for each individual animal. A higher AUC value indicates a greater overall pain relief (antinociceptive effect) over the 5-hour observation period.
Panel G and H:
Row Labels:
Animals: numerically represented, but placed by male and female groups each with experimental treatment variables ( Saline, CGX 0.02nmol, CGX 0.1nmol, CGX 0.5nmol)
BL = Baseline measurement (before surgery).
PostSx = 2 weeks post-surgery, immediately before treatment injection. 0.5, 1, 2, 4, 5 = Hours after treatment injection.
And for the units:
Units:
All values in Panel G (von Frey test) are reported as [e.g., withdrawal threshold in grams].
All values in Panel H (Hargreaves test) are reported as [e.g., paw withdrawal latency in seconds].
Panel I: Area under the curve analysis for mechanical withdrawal thresholds is presented in panel (G)
Panel J: Area under the curve analysis for thermal withdrawal latencies id presented in panel (H).
The AUC was calculated from the time of injection (time 0, which corresponds to the "PostSx" baseline) through the 5-hour time point for each individual animal. A higher AUC value indicates a greater overall pain relief (antinociceptive effect) over the 5-hour observation period.
figure 2:
Panel B:
Row Labels:
Animals: numerically represented, but placed by group ( Saline, CGX 0.02nmol, CGX 0.1nmol, CGX 0.5nmol)
Baseline = Baseline measurement (before surgery).
BL PostSx = 24 hours post-surgery, immediately before treatment injection. 0.5, 1, 2, 4, 5 = Hours after treatment injection.
Panel E:
Animals: numerically represented, but placed by group ( Saline, CGX 0.02nmol, CGX 0.1nmol, CGX 0.5nmol)
Baseline = Baseline measurement (before surgery).
BL PostSx = 2 weeks post-surgery, immediately before treatment injection. 0.5, 1, 2, 4, 5 = Hours after treatment injection.
And for the units:
Units:
All values in Panel B and E (von Frey test) are reported as [e.g., withdrawal threshold in grams].
Panel C and F: Area under the curve analysis for mechanical withdrawal thresholds is presented in panel (B and E)
The AUC was calculated from the time of injection (time 0, which corresponds to the "PostSx" baseline) through the 5-hour time point for each individual animal. A higher AUC value indicates a greater overall pain relief (antinociceptive effect) over the 5-hour observation period.
figure 3:
Panel B: quantification western blot images NTSR1 and NTSR2 expression calculated by percent. groups (Non Injected, Ctrl gRNA, NTSR2 gRNA)
Panel C:
Animals: numerically represented, but placed by group (Morphine-ctrl gRNA-ipsilateral, Morphine-ctrl gRNA, contralateral, 0.15 nM Cont-G-ctrl gRNA-ipsilateral, 0.15 nM Cont-G-ctrl gRNA, contralateral, Morphine-nstr2 gRNA-ipsilateral, Morphine-nstr2 gRNA, contralateral, 0.15 nM Cont-G-nstr2 gRNA-ipsilateral, 0.15 nM Cont-G-nstr2 gRNA, contralateral)
BL = Baseline measurement (before surgery).
BL PostSx = 2 weeks post-surgery, immediately before treatment injection. 1, 2, 4= Hours after treatment injection.
And for the units:
Units:
All values in Panel C (von Frey test) are reported as [e.g., withdrawal threshold in grams].
Panel D: Mechanical withdrawal thresholds before and 1 hour after morphine or CGX injections, in the same conditions as panel (C). 2 groups ( Ctrl gRNA, ntsr2 gRNA) treatments under groups (Mor ipsi BL Mor ipsi 1h CGX ipsi BL CGX ipsi 1h).
figure 4:
Panel B: Current–voltage relationships for total calcium currents. Peak currents were normalized to cell capacitance. units are Mb pot = membrane potential in (mV) (millivolts). groups are ( saline, CGX (10nM))
Panel C: peak current density (pA/pF) measured at +10 mV for CGX-treated neurons. groups (saline, CGX 10nM). units in (mV) (millivolts)
Panel E: Current–voltage relationships for R-type calcium currents are shown in. Peak currents were normalized to cell capacitance.units are Mb pot = membrane potential in (mV) (millivolts). groups are ( saline, CGX (10nM))
Panel F: peak current density (pA/pF) measured at +10 mV for CGX-treated neurons. groups (saline, CGX 10nM). units in (mV) (millivolts)
figure 5:
Panel B: quantification western blot images Cav2.3 and Cav2.2 expression calculated by percent. groups (Ctrl gRNA, Cav2.3 gRNA )
Panel C:
Animals: numerically represented, but placed by group (Morphine-ctrl gRNA-ipsilateral, Morphine-ctrl gRNA, contralateral, 0.15 nM Cont-G-ctrl gRNA-ipsilateral, 0.15 nM Cont-G-ctrl gRNA, contralateral, Morphine-nstr2 gRNA-ipsilateral, Morphine-nstr2 gRNA, contralateral, 0.15 nM Cont-G-nstr2 gRNA-ipsilateral, 0.15 nM Cont-G-nstr2 gRNA, contralateral)
BL = Baseline measurement (before surgery).
BL PostSx = 2 weeks post-surgery, immediately before treatment injection. 1, 2, 4= Hours after treatment injection.
And for the units:
Units:
All values in Panel C (von Frey test) are reported as [e.g., withdrawal threshold in grams].
Panel D: Mechanical withdrawal thresholds before and 1 hour after morphine or CGX injections, in the same conditions as panel (C). 2 groups ( empty vector, Cav2.3 CRISPR) treatments under groups (Mor ipsi BL Mor ipsi 1h CGX ipsi BL CGX ipsi 1h).
Figure 6:
Panel B: Quantification of cells expressing ntsr2, cacna1e, or both mRNA . numbers represented by percent cells positive in y axis and group in x axis. groups (Ntsr2 cells, Cacna1e cells, Co-exp)
Panel C: Relative expression of NSTR2 and Cav2.3 mRNA depending on cells expressing one of the mRNAs. . relative expression percent in y axis, number represent %. groups (Cav2.3 pos cell expressing Ntsr2, Ntsr2 pos cell expressing Cav2.3)
Panel D: DRG cell surface, depending on NTR2 mRNA expression, CaV2.3 mRNA expression, or both. x axis represents values in cell surface units=uM squared. groups (CoExp, CaV2.3, NTR2, Total)
Panel F:Values represent mean amplitude and frequency of sEPSC after treatments. data shown in values of amplitude and frequency (hertz). variables (Ctrl, Contulakin-G, 10 nM).
Panel G: KCl-depolarization evoked CGRP release from spinal afferents in mice. Isolated mouse lumbar spinal cords were washed (90 minutes), 2 baselines were collected (10 minutes fraction each, BL1 and BL2), and CGX (1-1000 nM) was pretreated (Tx) and cotreated with KCl (90 mM). Supernatant was analyzed for immunoreactive CGRP (iCGRP) using ELISA. data represented in CGRP release from pg/ml/mg of spinal cord. groups ( Saline, CGX 1nM, CGX 10nM, CGX 1000nM).
Panel I and J: data represents the quantification of Western blot data for Cav2.3 by synaptic region. represented as expression a.u. (arbitrary units). groups ( Postsyn, Presyn).
Panel J: A bar graph with scatterplot represents the quantification of Western blot data for NTSR2 by synaptic region. represented as expression a.u. groups ( Postsyn, Presyn).
Code/software
primary software used to analyze and collect data:
microsoft excel
graphpad prism
ImageJ