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Toxins (Basel)
2018 Jan 15;101:. doi: 10.3390/toxins10010043.
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The Peptide PnPP-19, a Spider Toxin Derivative, Activates μ-Opioid Receptors and Modulates Calcium Channels.
Freitas ACN
,
Peigneur S
,
Macedo FHP
,
Menezes-Filho JE
,
Millns P
,
Medeiros LF
,
Arruda MA
,
Cruz J
,
Holliday ND
,
Tytgat J
,
Hathway G
,
de Lima ME
.
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The synthetic peptide PnPP-19 comprehends 19 amino acid residues and it represents part of the primary structure of the toxin δ-CNTX-Pn1c (PnTx2-6), isolated from the venom of the spider Phoneutria nigriventer. Behavioural tests suggest that PnPP-19 induces antinociception by activation of CB1, μ and δ opioid receptors. Since the peripheral and central antinociception induced by PnPP-19 involves opioid activation, the aim of this work was to identify whether this synthetic peptide could directly activate opioid receptors and investigate the subtype selectivity for μ-, δ- and/or κ-opioid receptors. Furthermore, we also studied the modulation of calcium influx driven by PnPP-19 in dorsal root ganglion neurons, and analyzed whether this modulation was opioid-mediated. PnPP-19 selectively activates μ-opioid receptors inducing indirectly inhibition of calcium channels and hereby impairing calcium influx in dorsal root ganglion (DRG) neurons. Interestingly, notwithstanding the activation of opioid receptors, PnPP-19 does not induce β-arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, among opioid receptors, selectively activates μ-opioid receptors. The lack of β-arrestin2 recruitment highlights its potential for the design of new improved opioid agonists.
Figure 1. (A) Shows representative current traces of agonist-gated currents evoked from oocytes expressing human μ-opioid receptor (hMOR) by 1 μM morphine and 1 or 10 μM PnPP-19. PnPP-19 could not activate human δ-opioid receptor (hDOR) (B) or human κ-opioid receptor (hKOR) (C) up to a concentration of 10 μM.
Figure 2. Representative current traces evoked from X. laevis oocytes co-expressing GIRK1/GIRK2 channels and RGS4 with hMOR. In addition, 1 μM naloxone inhibits the agonistic activity of PnPP-19.
Figure 3. Effect of PnPP-19 and morphine on calcium current density evoked in dorsal root ganglion (DRG) neurons. Calcium currents were evoked by depolarizing pulses to 10 mV (200 ms) from a holding potential of −90 mV in DRG neurons incubated with 1 μM morphine, 1 μM PnPP-19 or 10 μM naloxone. Control group: cells incubated only with external/bath solution. Group NLX + PnPP-19: cells were previously incubated for 30 min with 10 μM naloxone and then PnPP-19 was added reaching a final concentration of 1 μM. MOR: morphine and NLX: naloxone. Data shown are the means ± SEM (n = 8 cells, 5 animals). * p < 0.05 compared with control (one-way ANOVA + Bonferroni’s test).
Figure 4. Representative trace showing calcium influx (changes in 340:380 nm ratios) in a single DRGneuron stimulated by KCl before and after 5 min incubation with buffer or PnPP-19. A perfusion system was used to incubate DRG neurons with buffer for 1 min (1) followed by consecutive KCl (30 mM) stimulations (2). After that, cells were perfused with buffer (3) or PnPP-19 (1 μM) (4) for 5 min, and again depolarized by KCl (30 mM) at three different time points (2). Control cells incubated only with buffer after first set of KCl stimulations do not show a significant difference in calcium influx during the second set of stimulations (A); However, cells incubated with PnPP-19 display a decrease in calcium influx during the second set of KCl stimulations (B); As a negative control, PnPP-19 does not influence calcium influx on its own (C).
Figure 5. Effect of pre-incubation with PnPP-19 (1 μM) on KCl-evoked (30 mM) responses. The bars represent the percentage of the maximum amplitude response during the second set of KCl stimuli corresponding to the initial KCl stimulations (100%). The peak of response in each situation was calculated, and the amplitude was assessed by diminishing this value of the baseline. The baseline corresponds to the pre-incubation of cells with buffer, before any KCl stimulation. Data shown are the means ± SEM (n = 5). * p < 0.05 compared with KCl (30 mM) + Buffer (two-tailed t-test).
Figure 6. Recruitment of β-arrestin2 by activation of μ-opioid receptors. Stably transfected HEK293 cells coexpressing μ-opioid receptors and β-arrestin2 were pretreated for 60 min with DAMGO or PnPP-19 at the indicated concentrations. The group âDAMGO + PnPP-19â represents prior incubation of the cells with 10 μM of PnPP-19 for 30 min. β-arrestin2 recruitment was quantified by high content imaging complementation assay as described in Materials and Methods (n = 5).
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