Analgesic α-Conotoxins Vc1.1 and Rg1A Inhibit N-Type Calcium Channels in Rat Sensory Neurons via GABA_B Receptor Activation

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.

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.

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.

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.

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.