Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation

Abstract

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.

Figure 1.

α-Conotoxin Vc1.1 inhibits HVA calcium channels in rat DRG neurons. A, Superimposed traces of depolarization-activated whole-cell calcium channel currents recorded using 2 mm Ba²⁺ as the charge carrier, elicited by a voltage step from a holding potential of −50 to 0 mV in the absence (a) and presence (b) of 100 nm Vc1.1. B, Corresponding time course of inhibition of peak Ba²⁺ current amplitude by 100 nm Vc1.1. Application of Vc1.1 is indicated by the bar. C, Plot of the I–V relationship for peak Ba²⁺ currents in the presence (●) and absence (○) of Vc1.1 (≤ 100 nm) (n = 16). D, Concentration–response curve for inhibition of calcium channel currents in DRG neurons by Vc1.1. Data points represent mean ± SEM of normalized peak current amplitude (n = 8–25 cells per data point). Maximum inhibition was 42 ± 3% with an IC₅₀ of 1.7 nm.

Figure 2.

Vc1.1 inhibition of HVA calcium channel currents is use dependent. A, Representative time course of peak Ba²⁺ current amplitude recorded using a 15 ms step depolarization from a holding potential of −80 mV to a test potential of −10 mV at a frequency of either 0.05 or 0.1 Hz, as indicated, in the absence and presence of 100 nm Vc1.1. B, Bar graph of the relative inhibition of HVA calcium channel currents by 100 nm Vc1.1 during a 15 ms depolarization at a frequency of either 0.05 Hz (n = 24) or 0.1 Hz (n = 7 responsive cells of 7 tested), and a pulse duration of 150 ms and frequency of either 0.05 Hz (n = 36 responsive cells of 50 tested) or 0.1 Hz (n = 9 responsive cells of 12 tested).***p < 0.001 unpaired t test. C, Superimposed traces of depolarization-activated whole-cell Ca²⁺ channel currents recorded using 2 mm Ca²⁺as the charge carrier, elicited by a voltage step from a holding potential of −80 to 0 mV in the absence (a) and presence (b) of 100 nm Vc1.1.

Figure 3.

Vc1.1 post-translationally modified analogs and other α-conotoxins are without effect on HVA calcium channel currents. A, Representative time course of peak Ba²⁺ current amplitude in the presence of 100 nm RgIA. The holding potential was −80 mV and the test potential −10 mV. B, Representative time course of peak Ba²⁺ current amplitude in the presence of 1 μm vc1a. C, Bar graph of relative inhibition of HVA calcium channel currents by α-conotoxins RgIA, ImI, [A10L]PnIA, MII, BuIA, alkylated Vc1.1, Vc1.1, and analogs. Vc1.1 (1 μm) inhibited peak Ba²⁺ current amplitude in 25 of 30 cells tested. ***p = 0.0001 compared with control cells. Rg1A (100 nm) inhibited peak Ba²⁺ current amplitude in 11 of 14 cells tested. **p = 0.0016 compared with control cells. Numbers in parentheses reflect the number of cells.

Figure 4.

Vc1.1 inhibits N-type calcium channels in rat DRG neurons. A, Superimposed traces of depolarization-activated whole-cell Ba²⁺ currents under control conditions (a), in the presence of 200 nm ω-conotoxin CVID (b), and in the presence of CVID and 1 μm Vc1.1 (c). B, Representative time course of peak Ba²⁺ current amplitude in the presence of the selective N-type Ca²⁺ channel inhibitor ω-conotoxin CVID followed by application of Vc1.1. The letters a–c indicate the time points at which the superimposed currents were obtained. C, Bar graph of relative inhibition of HVA calcium channel currents by 200 nm CVID alone, after application of 1 μm Vc1.1 in the presence of CVID, in the presence of Vc1.1 alone, and after application of CVID in the presence Vc1.1. Numbers in parentheses reflect the number of cells.

Figure 5.

Vc1.1 does not inhibit Ba²⁺ currents through recombinant N- and P/Q-type channels expressed in Xenopus oocytes. A, Superimposed current traces from an oocyte expressing Ca_v2.2 α_1B-d/β₃/α₂δ₁ (N-type) calcium channels under control conditions (a) and in the presence of 1 μm Vc1.1 (b). B, Superimposed currents from an oocyte expressing Ca_v2.1 α_1E/β₃/α₂δ₁ (P/Q-type) calcium channels in the absence (control) (a) and presence (b) of 1 μm Vc1.1. C, Bar graph of relative current amplitude obtained for expressed N- and P/Q-type calcium channels in the presence of 1 μm Vc1.1. Numbers in parentheses reflect the number of cells.

Figure 6.

Vc1.1 inhibition of N-type calcium channel currents is mediated by G-protein-coupled receptor and tyrosine kinase activation. A(i), Superimposed traces in the absence (a) and presence (b) of 1 μm Vc1.1 in cells incubated overnight in PTX (3 μg/ml). Cells were recorded under continual perfusion of PTX (200 ng/ml). (ii), Corresponding time course. B(i), Superimposed traces in the absence (a) and presence (b) of Vc1.1 (1 μm) in cells recorded with pp60c-src peptide (521-533) inhibitory peptide (100 μm) included in the recording pipette. (ii), Corresponding time course. C, Bar graph of relative inhibition of HVA calcium channel currents by Vc1.1 (1 μm) in regular recording solution (GTP), when a prepulse to +80 mV (+PP) was applied 10 ms before the test pulse, when GTP was replaced with GDPβS, after overnight incubation in PTX, or when pp60c-src inhibitory peptide was included in the pipette recording solution. ***p = 0.0001 compared with control. Numbers in parentheses reflect the number of cells.

Figure 7.

GABA_B receptor activation mediates Vc1.1 inhibition of N-type calcium channel currents in rat DRG neurons. A(i), Superimposed traces of depolarization-activated whole-cell Ba²⁺ currents in the presence of the GABA_B inhibitor phaclofen (50 μm) followed by application of 100 nm Vc1.1. (ii), Corresponding time course. Numbers on the plot indicate which points were used for sample traces in A(i). B(i), Superimposed traces of depolarization-activated whole-cell Ba²⁺ currents in the presence of another GABA_B inhibitor CGP55845 (1 μm) (a), in the presence of CGP55845 and Vc1.1 (100 nm) (b), and in the presence of Vc1.1 alone (100 nm) (c). (ii), Corresponding time course. Numbers on the plot indicate which points were used for sample traces in B(i). C(i), Superimposed traces of depolarization-activated whole-cell Ba²⁺ currents in the absence (a), in the presence of the GABA_B agonist baclofen (30 μm) (b), and in the presence of 100 nm Vc1.1 (c). (ii), Corresponding time course. Numbers on the plot indicate which points were used for sample traces in C(i).