α-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 α9α10 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 α-conotoxins tested. Vc1.1 inhibited the ω-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing CaV2.2, CaV2.1, CaV2.3, or CaV1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPβS, 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 GABAB 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 CaV2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which α-conotoxins Vc1.1 and Rg1A modulate native N-type (CaV2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABAB receptors.
Conotoxins are small peptides derived from the venom of the Conus genus of predatory marine snails (Terlau and Olivera, 2004). Many conotoxins are highly selective antagonists of a diverse range of mammalian ion channels and receptors associated with pain pathways. Thus, conotoxins are attractive as drug leads for treating neuropathic pain. Previous studies have demonstrated the analgesic potential of several different classes of conotoxins (Adams et al., 1999; Livett et al., 2004) and, most recently, antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) have begun to show promise for the treatment of neuropathic pain (Sandall et al., 2003; Lang et al., 2005; Satkunanathan et al., 2005; Vincler et al., 2006). The α-conotoxins competitively inhibit neuronal-type nAChRs (Dutton and Craik, 2001) with varying degrees of selectivity for different nAChR subtypes (McIntosh et al., 1999).
α-Conotoxin Vc1.1, which was first discovered using a PCR screen of cDNAs from the venom ducts of Conus victoriae (Sandall et al., 2003), contains two residues, Pro6 and Glu14, that are post-translationally modified to hydroxyproline and γ-carboxyglutamate, respectively, in the native peptide, designated vc1a (Jakubowski et al., 2004). Synthetic Vc1.1 lacking these two modifications is a competitive antagonist of neuronal nAChRs in bovine adrenal chromaffin cells (Clark et al., 2006) and is potent at recombinant α9α10 nAChRs expressed in Xenopus oocytes (Vincler et al., 2006; Nevin et al., 2007). Furthermore, Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14γ]Vc1.1 have been reported to be equally potent at inhibiting ACh-evoked currents mediated by α9α10 nAChRs (Nevin et al., 2007). Vc1.1 also antagonizes the nicotine-induced increase in axonal excitability of unmyelinated C-fiber axons in isolated segments of peripheral human nerves (Lang et al., 2005). Blockade of nAChRs on unmyelinated peripheral nerve fibers may have an analgesic effect on unmyelinated sympathetic and/or sensory axons. Interestingly, synthetic vc1a, which incorporates the modified residues, has been reported to inhibit α9α10 (Nevin et al., 2007) but not α3-containing (Clark et al., 2006) nAChRs expressed in Xenopus oocytes and is inactive in two rat neuropathic pain assays (Livett et al., 2006). In contrast, Vc1.1 alleviates neuropathic pain in three rat models of human neuropathic pain and accelerates the functional recovery of injured neurons (Satkunanathan et al., 2005). α-Conotoxin Rg1A from Conus regius, which also selectively inhibits α9α10 nAChRs (Ellison et al., 2006), has similarly been shown to significantly reduce chronic constriction nerve injury-induced hyperalgesia in rats (Vincler et al., 2006; Vincler and McIntosh, 2007). Thus, although Vc1.1 is currently under development as a treatment for neuropathic pain (Livett et al., 2004, 2006), its specific receptor target has not been unequivocally identified. We show here that Vc1.1 and Rg1A act by an unexpected mechanism not reported previously for any conotoxin.
Materials and Methods
DRG neuron preparation.
DRG neurons were enzymatically dissociated from ganglia of 2- to 30-d-old Wistar rats according to standard protocols. Rats were killed by cervical dislocation as approved by the University of Queensland Animal Ethics Committee, the spinal column was hemi-segmented, and the spinal cord was removed. Ganglia were removed and rinsed in cold HBSS (MultiCell). They were minced and incubated in 1 mg/ml collagenase (type 2; 405 U/mg) (Worthington Biochemical) in HBSS at 37°C for ∼30 min. After incubation, ganglia were rinsed three times with warm (37°C) DMEM (Invitrogen) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin, and gently triturated with a fire-polished Pasteur pipette. Cells were plated on glass coverslips, incubated at 37°C in 95% O2/5% CO2, and used within 4–48 h.
The external recording solution for rat DRG neurons contained the following (in mm): 150 tetraethylammonium (TEA)-Cl, 2 BaCl2, 10 d-glucose, and 10 HEPES, pH 7.3–7.4. Recording electrodes were filled with an internal solution containing the following (in mm): 140 CsCl, 1 MgCl2, 5 MgATP, 0.1 Na-GTP, 5 bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA)-Cs4, and 10 HEPES, pH 7.3–7.4, with CsOH, and had resistances of 1–2.5 MΩ. Membrane currents were recorded using the whole-cell configuration of the patch-clamp technique with an Axopatch 200B amplifier (Molecular Devices). Neurons were voltage clamped at a holding potential of −90 mV and Ba2+ currents were elicited by step depolarizations, first to −40 mV for 200 ms [for low-voltage-activated (LVA) currents] and then to −10 mV, from a holding potential of −50 mV, for 200 ms [for high-voltage-activated (HVA) currents] at 10 s intervals. In a series of experiments, BaCl2 was replaced by CaCl2. A voltage protocol using step depolarizations from −70 to 0 mV was used when examining HVA currents alone. Test potentials 150 ms in duration were applied every 20 s. To construct current–voltage (I–V) relationships, neurons were voltage clamped at −100 mV and voltage stepped from −80 mV to +50 mV in 10 mV increments. Voltage-dependent inhibition was assessed by applying a prepulse to +80 mV for 20 ms in duration before the test pulse. Leak and capacitative currents were subtracted using a −P/4 pulse protocol. Currents were generated by a computer using pClamp 9.2 software (Molecular Devices), filtered at 2 kHz, and sampled at 8 kHz by the Digidata 1322A (Molecular Devices). Sampled data were stored digitally on a computer for further analysis.
Oocyte preparation and recordings.
Stage V–VI oocytes from Xenopus laevis frogs were injected with either cRNA or cDNA encoding N- or P/Q-type voltage-gated Ca2+ channels (VGCCs), respectively. N-type channels were examined in oocytes injected with 50 nl of solution containing a mixture of cRNAs encoding rat CaV2.2 α1B-b subunit (2.5–5 ng/cell) and rat β3 subunit (2.5–5 ng/cell) (cDNAs provided by Dr. D. Lipscombe, Brown University, Providence, RI), with or without rabbit α2δ1 subunit (2.5–5 ng/cell) (cDNA provided by Dr. F. Hofmann and Dr. N. Klugbauer, Technische Universität München, Munich, Germany). L-type channels were examined in oocytes coinjected with rabbit Cav1.2 α1C subunit (5 ng/cell) (cDNA provided by Dr. G. Zamponi, University of Calgary, Calgary, Alberta, Canada) and auxiliary subunits β3 and α2δ1 (see above). For experiments examining P/Q-type and R-type VGCCs, the oocyte nucleus was injected first with cDNA encoding the rabbit CaV2.1 α1A (5 ng/cell) (cDNA provided by Dr. G. Zamponi) or rat Cav2.3 α1E (4.5 ng/cell) (cDNA provided by Dr. G. Zamponi) subunit, respectively, followed by cytoplasmic injection of auxiliary subunits β3 and α2δ1 (see above). After injection, oocytes were kept at 18°C for 3–6 d before use.
Before recording, oocytes were injected with 25 nl of 50 mm BAPTA to eliminate the endogenous Ca2+-activated chloride conductance. Depolarization-activated Ca2+ channel currents were recorded using Ba2+ as a charge carrier and a two-electrode virtual ground voltage-clamp circuit with a GeneClamp 500B amplifier (Molecular Devices). The external bath solution contained the following (in mm): 85 TEA-OH, 5 BaCl2, 5 KCl, and 10 HEPES, pH 7.4, with methanesulfonic acid. During recording, oocytes were constantly perfused with bath solution using a gravity-fed perfusion system at a flow rate of ∼1.5 ml/min. Electrodes were filled with a solution containing 3 m KCl (0.5–1.0 MΩ). Oocytes were voltage clamped at a holding potential of −80 mV and membrane currents were elicited by 200 ms step depolarization to 0 mV (for CaV2.2) or +20 mV (for CaV2.1) applied every 10 s. To construct I–V relationships, cells were voltage-clamped at −80 mV and then stepped from −80 mV to +50 mV in 10 mV increments. Data were low-pass filtered at 2 kHz, digitized at 10 kHz, leak subtracted online using a −P/4 protocol, and analyzed off-line.
Data were analyzed off-line using pClamp 9.2 software (Molecular Devices). Peak Ba2+ current amplitude in response to a depolarizing pulse was calculated and plotted against time to monitor stability of the recording. Generally, Ba2+ current reached steady state within 2–5 min of initial recording, and any recordings that did not reach steady state were discarded. Drugs were applied via perfusion in the bath solution. Current amplitudes obtained in the presence of the drug were normalized by dividing by the current amplitude obtained under control conditions. Concentration–response relationships were obtained using GraphPad Prism software by plotting normalized current amplitude as a function of drug concentration and were fitted using a logistic equation. Numerical data are presented as mean ± SEM.
Drugs and chemicals.
CGP55845 (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid, CGP54626 [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride], and pp60c-src peptide (521-533) corresponding to its C-terminal regulatory domain were purchased from Tocris Bioscience. The peptide binds pp60c-src at the Src homology 2 domain suppressing its tyrosine kinase activity (Roussel et al., 1991). Phaclofen (3-amino-2-(4-chlorophenyl)propylphosphonic acid was from BIOMOL. All other drugs were obtained from Sigma-Aldrich and were prepared as concentrated stock solutions which were then diluted in bath solution and applied via perfusion. The ω-conotoxins CVID and MVIIA were prepared as described previously (Lewis et al., 2000). α-Conotoxins Vc1.1, [P6O]Vc1.1, [E14γ]Vc1.1, vc1a, RgIA, ImI, MII, BuIA and [A10L]PnIA, were synthesized as reported previously (Gehrmann et al., 1999; Hogg et al., 1999; Blanchfield et al., 2003; Clark et al., 2006, 2008; Jin et al., 2007) and provided as a stock concentration in H2O of ∼1 mm and used at a final concentration of 0.1 nm to 1 μm. Reduced Vc1.1 was prepared by incubation with dithiothreitol (2 mm; 1 h; room temperature) or alkylated with iodoacetamide in 0.1 m ammonium acetate buffer.
α9α10 nAChR-selective conotoxins, Vc1.1 and Rg1A, inhibit voltage-gated Ca2+ channel currents in rat DRG neurons
In an attempt to account for α-conotoxin Vc1.1-induced analgesia and to elucidate the receptor target and mechanism of action, the effects of the conotoxins, Vc1.1 and RgIA, were investigated on voltage-gated Ca2+ channel currents in rat DRG neurons. Whole-cell Ca2+ channel currents of rat DRG neurons are mediated by both LVA and HVA calcium channels (Scroggs and Fox, 1992). The effect of Vc1.1 on Ca2+ channel currents recorded from neonatal (2- to 10-d-old) rat DRG neurons was initially assessed using a two-step protocol (see Materials and Methods). Vc1.1 (1 μm) had no effect on LVA Ca2+ channel current amplitude elicited by voltage step from −90 to −40 mV (97 ± 12% of control, n = 10). In the same cells, Vc1.1 inhibited HVA Ca2+ channel currents elicited by a 150 ms voltage step to −10 mV to 56.5 ± 4.4% of control (n = 10) (Fig. 1A,B). Vc1.1 only inhibited VGCC currents evoked by step depolarization positive of −40 mV, demonstrating the lack of effect of Vc1.1 on LVA Ca2+ channel currents (Fig. 1C). The effect of Vc1.1 on HVA Ca2+ channel currents was therefore assessed using a 150 ms step depolarization from a holding potential of either −70 or −90 mV to the test potential of either −10 or 0 mV at a frequency of 0.05 Hz, with 2 mm Ba2+ as the charge carrier. Vc1.1 inhibition of HVA Ca2+ channel currents developed with a delay of ∼2 min and maximum inhibition occurred ∼7 min after the beginning of the response, independent of the concentration of Vc1.1 (n = 74). Inhibition of HVA Ca2+ channel currents by Vc1.1 was concentration dependent and the mean concentration–response data gave an IC50 of 1.7 nm, with maximum inhibition to 57.8 ± 3.8% of control occurring at 1 μm (n = 25) (Fig. 1D).
Vc1.1 (100 nm) inhibited VGCC currents in >75% of DRG neurons (120 of 159) tested, including small (<20 μm), medium (20–40 μm), and large (>40 μm) neurons. Only a subset of primary sensory DRG neurons are capsaicin sensitive (Szallasi and Blumberg, 1999). There was no correlation between cell size or capsaicin sensitivity and responsiveness to Vc1.1: 88% of small cells (n = 17), 71% of medium cells (n = 51), and 76% of large cells (n = 91) responded to ≥10 nm Vc1.1; 5 of 8 capsaicin-sensitive cells responded to Vc1.1 and 6 of 9 capsaicin-insensitive cells responded to Vc1.1. Vc1.1 (100 nm) also inhibited VGCC currents in 14 of 25 juvenile (11- to 20-d-old) and 4 of 6 adult (>28-d-old) rats (67.5 ± 3.3% and 69.4 ± 3.2% of control, respectively).
Vc1.1 inhibition of VGCC currents was not reversed during washout. However, reapplication of Vc1.1 produced an equivalent inhibition of HVA Ca2+ channel currents in other DRG neurons on the same coverslip (n = 5 sets), suggesting that depolarization-induced Ca2+ channel activation is required for inhibition by Vc1.1.
To further assess the dependence of Vc1.1 inhibition on activation of HVA Ca2+ channel currents, we varied the frequency and duration of the depolarizing pulse in the presence of Vc1.1 (Fig. 2). When the depolarization pulse was shortened to 15 ms with the frequency maintained at 0.05 Hz, 100 nm Vc1.1 inhibited the current to only 88.4 ± 3.3% of control in all cells tested (n = 24). However, when the frequency of the 15 ms pulse was increased to 0.1 Hz, the current was reduced to 51.1 ± 5.6% of control in all cells tested (n = 7) (Fig. 2A,B). Figure 2B also shows the relative inhibition of Ba2+ currents by Vc1.1, using the standard depolarizing pulse duration (150 ms) at both 0.05 Hz and 0.1 Hz. The action of Vc1.1 does not depend on a rise in intracellular divalent cation, because the inhibition was also observed with 2 mm Ca2+ as the charge carrier. In 9 of 12 cells, Vc1.1 (100 nm) inhibited the HVA Ca2+ current amplitude to 61.4 ± 5.2% of control and was not reversible on washout (>30 min) (Fig. 2C). Furthermore, in fura-2-loaded DRG neurons, bath application of 1 μm Vc1.1 alone failed to elicit any increase in intracellular Ca2+ concentration (n = 14, data not shown).
Application of α-conotoxin Rg1A (100 nm) also significantly inhibited HVA Ca2+ channel currents in rat DRG neurons (11 of 14 responding cells) to 60.6 ± 4.5% of control (Fig. 3A).
Post-translationally modified analogs of Vc1.1 and other α-conotoxins do not inhibit HVA Ca2+ channel currents in DRG neurons
The effects of post-translationally modified analogs of Vc1.1 were examined on HVA Ca2+ channel currents from rat DRG neurons under the same conditions as Vc1.1. The native peptide, vc1a, did not inhibit HVA Ca2+ channel currents (100.0 ± 4.8% of control, n = 9); however, the same cells responded to Vc1.1 after washout of vc1a (Fig. 3B,C). [P6O]Vc1.1 had no effect on HVA Ca2+ channel currents (103.7 ± 7.2% of control, n = 9) (Fig. 3C), whereas four of five cells from this test group subsequently responded to Vc1.1 after washout of [P6O]Vc1.1. [E14γ]Vc1.1 was also without effect (110.1 ± 9.2% of control, n = 7) (Fig. 3C), but after its removal, all cells of this group tested with Vc1.1 showed a response (n = 6). Both reduced Vc1.1 and alkylated Vc1.1 (1 μm) were without effect on HVA Ca2+ channel currents, whereby the peak current amplitude was 98.8 ± 7.1% of control (n = 10) and 104.9 ± 11.4% of control (n = 3), respectively (Fig. 3C).
The effects of other 4/7 α-conotoxins (MII and [A10L]PnIa) as well as the 4/3 α-conotoxin ImI, and the 4/4 α-conotoxin BuIA were examined to ascertain whether inhibition of HVA Ca2+ channel currents was a common characteristic of α-conotoxins. α-Conotoxins ImI, MII, BuIA, and [A10L]PnIA applied at a concentration of either 100 nm or 1 μm did not inhibit HVA Ca2+ channel currents in DRG neurons (ImI, 95.5 ± 5.1% of control, n = 7; MII, 93.2 ± 1.6% of control, n = 3; BuIA, 97.5 ± 7.9% of control, n = 8; [A10L]PnIA, 91.0 ± 4.1% of control, n = 10) (Fig. 3C). Another conopeptide structurally related to the 4/7 α-conotoxins, ρ-TIA, an allosteric α1-adrenoreceptor antagonist (Sharpe et al., 2003), also failed to inhibit HVA Ca2+ channel currents in DRG neurons when bath applied at 100 nm (n = 6, data not shown).
Vc1.1 inhibits the N-type component of HVA Ca2+ channel currents in DRG neurons
The selective N-type Ca2+ channel blockers, ω-conotoxins CVID and MVIIA, were applied to determine which component of the whole-cell HVA Ca2+ channel currents Vc1.1 inhibited in DRG neurons. Bath application of 200 nm CVID reduced peak Ca2+ channel current amplitude to 54.4 ± 4.8% of control (n = 6). Application of 1 μm Vc1.1 in the presence of CVID (200 nm) produced no further reduction of the HVA Ca2+ channel current amplitude (51.7 ± 5.1% of control, n = 6) (Fig. 4A–C). Figure 4C also shows the result of the converse experiment, that is, the average response of cells to CVID (200 nm) after Vc1.1 (≥100 nm) application. Similar results were obtained with bath application of 100 nm ω-conotoxin MVIIA, with 51.6 ± 5.6% of the current remaining after application of MVIIA and 50.4 ± 0.1% remaining in the presence of MVIIA and 100 nm Vc1.1 (n = 5) (data not shown).These data suggest that Vc1.1 inhibits the N-type Ca2+ channel in DRG neurons.
Action of Vc1.1 on expressed CaV2.1 and CaV2.2 channels in oocytes
To assess whether Vc1.1 directly inhibits HVA Ca2+ channels, the α subunits of CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels were expressed in Xenopus oocytes together with the auxiliary subunits β3/α2δ1 in the ratio 1:1:1. Bath application of 1 μm Vc1.1 did not significantly inhibit the Ba2+ current amplitude for oocytes expressing CaV2.2 channels (91.3 ± 2.2% of control, n = 8) (Fig. 5A,C). Analogous to CaV2.2-expressing oocytes, 1 μm Vc1.1 did not inhibit Ba2+ currents mediated by CaV2.1 channels (88.1 ± 8.2% of control, n = 4) (Fig. 5B,C). Raising the Vc1.1 concentration to 10 μm similarly failed to inhibit the Ba2+ currents mediated by either CaV2.1 or CaV2.2 channels expressed in oocytes. A similar lack of effect of Vc1.1 on CaV2.3 (R-type) and CaV1.2 (L-type) channels expressed in Xenopus oocytes was also observed (data not shown). To investigate whether the auxiliary subunits influence the sensitivity of recombinant Ca2+ channels to Vc1.1 (Yasuda et al., 2004), oocytes were injected with the subunit combination CaV2.2/β3 in a 1:1 ratio. Although cells expressing CaV2.2/β3 elicited smaller Ca2+ channel currents than those expressed by CaV2.2/β3/α2δ1 channels, the Ba2+ current amplitude was not appreciably changed (88 ± 6.5% of control, n = 3) in the presence of 1 μm Vc1.1 (Fig. 5C). Together, these data indicate that Vc1.1 does not interact with either CaV2.1 (P/Q-type) or CaV2.2 (N-type) channels directly, and that Vc1.1 modulation of HVA Ca2+ channel currents occurs via an indirect mechanism.
Vc1.1 inhibits HVA Ca2+ channel currents via a G-protein-coupled receptor mechanism and src tyrosine kinase
The role of G-proteins in mediating Vc1.1 inhibition of HVA Ca2+ channel currents in DRG neurons was examined by replacing GTP with GDPβS, a nonhydrolyzable GDP analog, in the intracellular recording solution. GDPβS antagonized the effect of Vc1.1 on Ca2+ channel currents, with 96.8 ± 4.5% of current remaining after addition of 100 nm Vc1.1 in the presence of GDPβS (n = 15) (Fig. 6C).
These results suggest that Vc1.1 inhibits N-type Ca2+ channel currents in DRG neurons via a G-protein-coupled receptor (GPCR) mechanism. Vc1.1 inhibition also persisted after application of depolarizing prepulses to +80 mV, suggesting a voltage-independent inhibitory pathway (n = 7) (Fig. 6C). Incubation of cells in medium containing 3 μg/ml pertussis toxin (PTX) for 24 h antagonized the inhibition of VGCC currents by Vc1.1 (99.9 ± 4.5% of control, n = 11) (Fig. 6A,C). Together, these results suggest that the mechanism by which Vc1.1 inhibits HVA Ca2+ channel currents involves a PTX-sensitive GPCR of the Gαi/o subfamily.
Nonreceptor tyrosine kinases have been implicated in voltage-independent inhibition of N-type Ca2+ channel currents initiated by activation of GPCR pathways (Diversé-Pierluissi et al., 1997; Strock and Diversé-Pierluissi, 2004; Raingo et al., 2007). We therefore tested the ability of Vc1.1 to inhibit HVA Ca2+ channel currents when a selective peptide inhibitor of pp60c-src tyrosine kinase (Roussel et al., 1991) (100 μm) was included in the intracellular recording solution. Vc1.1 had no effect on HVA Ca2+ channel currents in the presence of the pp60c-src peptide (101.39 ± 6.9% of control, n = 10) (Fig. 6B,C).
Vc1.1 inhibits HVA Ca2+ channel currents in DRG neurons via GABAB receptor
To investigate further the identity of the primary target for Vc1.1, a number of different receptor antagonists were tested for their ability to antagonize the effect of Vc1.1 (Table 1). Antagonists of neuronal nAChRs, mecamylamine and hexamethonium, and desensitizing concentrations of ACh were bath applied ∼5 min before application of Vc1.1 and in all cases failed to antagonize the inhibition of N-type VGCC currents in DRG neurons by Vc1.1. Similarly, the muscarinic acetylcholine receptor antagonist, atropine, the α1- and α2-adrenergic receptor antagonists, prazosin and yohimbine, respectively, the μ-opioid receptor antagonist, naloxone, and the GABAA receptor antagonist, bicuculline, all failed to antagonize the effect of Vc1.1 in the majority of cells tested.
Numerous studies have shown that activation of GABAB receptors leads to inhibition of N-type Ca2+ channel currents in central and peripheral neurons (Menon-Johansson et al., 1993; Guyon and Leresche, 1995; Fujikawa et al., 1997; Harayama et al., 1998; Richman et al., 2005; Castro et al., 2007; Raingo et al., 2007), but conotoxins have not previously been contemplated to act on this pathway. The inhibition of N-type Ca2+ channel currents after activation of GABAB receptors has been shown to occur in both a voltage-dependent and a voltage-independent manner, to be PTX-sensitive, and to involve c-Src (Richman et al., 2005; Raingo et al., 2007). Several GABAB receptor antagonists were tested for their ability to antagonize Vc1.1 inhibition of HVA Ca2+ channel currents in DRG neurons. Application of 1 μm Vc1.1 in the presence of the GABAB receptor antagonists phaclofen (50 μm) and CGP55485 (1 μm) did not significantly reduce the HVA Ca2+ channel current in DRG neurons [95.5 ± 4.4% (n = 9 and 93.2 ± 4.5% (n = 14) of control, respectively] (Fig. 7A,B). RgIA (100 nm) also failed to inhibit HVA Ca2+ channel current in the presence of CGP55485 (92.5 ± 3.5% of control, n = 5) (data not shown). Together, these data indicate that Vc1.1 and Rg1A inhibit HVA Ca2+ channel currents via activation of the GABAB receptor in DRG neurons.
The GABAB receptor agonist baclofen has previously been shown to inhibit HVA Ca2+ channel currents in neonatal rat DRG neurons (Dolphin and Scott, 1987, 1990; Menon-Johansson et al., 1993; Fujikawa et al., 1997). We examined whether the effects of Vc1.1 and baclofen were additive in inhibition of the HVA Ca2+ channel current. The inhibition produced after addition of 10–30 μm baclofen (63.6 ± 10.6% of control, n = 4) was not significantly greater than that produced by 100 nm Vc1.1 alone (61.2 ± 15.9% of control, n = 4) in the same cells. Similarly, application of Vc1.1 (100 nm) after addition of 30 μm baclofen did not further inhibit HVA Ba2+ current amplitude (63.6 ± 7.2% of control, n = 5) compared with baclofen alone (64.6 ± 6.6% of control, n = 5) (Fig. 7C). Under the present recording conditions (150 ms depolarizing pulse to −10 mV applied at a frequency of 0.05 Hz), the time to maximum inhibition (9.3 ± 1.2 min) and reversal (<5% after 10 min washout) by 30 μm baclofen (n = 11) was significantly slower than when the depolarizing pulse was shortened to 15 ms or the frequency of depolarization was increased to 0.1 Hz (time to maximum inhibition occurred in 2.8 ± 0.9 min and complete recovery within 5 min after washout of baclofen, n = 5).
In subsequent experiments, we observed that in ∼70% of oocytes expressing recombinant CaV2.2 (N-type) but not CaV2.1 (P/Q-type), CaV2.3 (R-type), or CaV1.2 (L-type) calcium channels, application of baclofen (100 μm) inhibited Ba2+ currents (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). We therefore tested the effect of α-conotoxins Vc1.1 and Rg1A in these oocytes. Both Vc1.1 and Rg1A inhibited Ba2+ currents through recombinant CaV2.2 channels in baclofen-responding oocytes (n ≥ 28). Preincubation of oocytes with the selective GABAB receptor antagonist CGP55845 (1 μm) abolished the inhibition of Ba2+ currents observed with 1 μm Vc1.1 (n = 4) (supplemental Fig. 2, available at www.jneurosci.org as supplemental material).
To date, the α-conotoxins have been classified as competitive nAChR antagonists (Dutton and Craik, 2001; Livett et al., 2006). The present study is the first to identify the N-type calcium channel of mammalian DRG neurons as a target of the α-conotoxins Vc1.1 and RgIA. To the best of our knowledge, this study is also the first to describe conotoxin modulation of an ion channel via a G-protein receptor-activated pathway.
Vc1.1 and Rg1A are of great interest as analgesic drugs because of their effectiveness in peripheral nerve-injured rats (Satkunanathan et al., 2005; Vincler et al., 2006) Although these peptides have been shown to be potent antagonists of α9α10 nAChRs, post-translationally modified analogs of Vc1.1, [P6O]Vc1.1, and vc1a are ineffective in assays of neuropathic pain (Livett et al., 2006; Nevin et al., 2007). However, they are equipotent to Vc1.1 as α9α10 nAChRs antagonists, indicating that this nAChR is not the therapeutic target of Vc1.1 (Nevin et al., 2007).
N-type calcium channels are an appealing target of Vc1.1 because they are well known to play a central role in the detection and transmission of nociceptive stimuli in DRG neurons (Altier and Zamponi, 2004). Several studies have highlighted the importance of N-type calcium channels in neuropathic pain: N-type α1B channel knock-out mice have a decreased response to neuropathic pain (Hatakeyama et al., 2001; Kim et al., 2001; Saegusa et al., 2001), there is an upregulation of N-type α1B as well as α2δ subunits in rat nerve injury models (Abe et al., 2002; Cizkova et al., 2002; Yokoyama et al., 2003), and currently used treatments, or treatments being developed, for pain relief include direct (ω-conotoxin MVIIA, also known as Prialt) and indirect (morphine) inhibitors of N-type channels (McGivern, 2006).
HVA Ca2+ channel currents of rat DRG neurons include contributions from N-, P/Q-, L-, and R-type calcium channels (Scroggs and Fox, 1992). The present study indicates that Vc1.1 targets solely the N-type component of the whole-cell Ca2+ current, because it had no effect in the presence of the N-type Ca2+ channel blockers, ω-conotoxins CVID and MVIIA. Vc1.1 inhibition of N-type Ca2+ channel current was irreversible after washout of up to 30 min, but subsequent cells patched from the same coverslip were as sensitive to Vc1.1 as the initial cell exposed to Vc1.1. This suggests that Vc1.1 inhibition of N-type current is dependent on membrane depolarization. Consistent with this hypothesis, Vc1.1 had no effect on HVA Ca2+ currents in DRG neurons when the duration of the depolarization pulse was shortened to one-tenth of its initial duration with the frequency maintained at 0.05 Hz. Vc1.1 inhibition was only observed with this short depolarizing pulse when the frequency was increased to 0.1 Hz.
DRG neurons have been shown to express a splice variant CaV2.2 that differs at exon 37 (Bell et al., 2004) from the peripheral splice variant we expressed, but extensive studies reported here indicate that the reason Vc1.1 is ineffective on recombinant N-type Ca2+ channels is that it is acting via a G-protein receptor-activated pathway. Our results suggest that the GPCR responsible is the GABAB receptor: first, Vc1.1 inhibition of N-type Ca2+ current was antagonized by selective GABAB receptor antagonists, and second, similar to previous studies of the GABAB receptor agonist baclofen in rat or chick DRG neurons (Dolphin and Scott, 1987; Richman and Diversé-Pierluissi, 2004; Raingo et al., 2007), Vc1.1 inhibition of N-type Ca2+ currents in rat DRG neurons was antagonized when GTP was replaced in the patch pipette with GDPβS, when the cells were incubated overnight with PTX, and when c-Src tyrosine kinase activity was inhibited. Activation of the GABAB receptor has been shown to inhibit N-type Ca2+ channel current by voltage-dependent (relieved by a strong depolarizing prepulse) and voltage-independent pathways, and to be readily reversible after washout of baclofen (Richman and Diversé-Pierluissi, 2004; Raingo et al., 2007). No relief of Vc1.1 inhibition was observed after depolarizing prepulses to +80 mV. Although baclofen inhibition during prolonged depolarizations (150 ms, 0.05 Hz) was also irreversible, baclofen inhibition of N-type Ca2+ current during short depolarization pulses (15 ms, 0.05 Hz) was rapidly reversible after washout of baclofen (our unpublished observations). These results suggest that Vc1.1 does not inhibit the N-type current via the fast, Gβγ-mediated, membrane-delimited, voltage-dependent pathway associated with GABAB receptor activation. Rather, it activates a pathway that remains to be fully elucidated but involves pp60c-src tyrosine kinase and leads to a maintained inhibition of the N-type Ca2+ channel current, possibly as a result of persistent binding of Vc1.1 to the GABAB receptor. Alternatively Vc1.1 may interact differently with the GABAB receptor than baclofen, which binds to the venus fly trap motif of the GABAB1 subunit, leading to its closure and activation of the receptor (for review, see Emson, 2007).
Vc1.1 was without effect on recombinant CaV2.1 (P/Q-type), CaV1.2 (L-type), and CaV2.3 (R-type) channels expressed in Xenopus oocytes. However, in subsequent experiments we found that baclofen activation of endogenous GABAB receptors in batches of Xenopus oocytes mediates inhibition of Ba2+ current through recombinant CaV2.2 (N-type) channels. In oocytes expressing endogenous GABAB receptors, α-conotoxins Vc1.1 and Rg1A also inhibit recombinant CaV2.2 channels via agonist activation of a GABAB receptor-signaling pathway in Xenopus oocytes (Yang et al., 2001).
The characteristics of the G-protein-induced inhibition of voltage-dependent N-type calcium channels by Vc1.1 and Rg1A activation of GABAB receptors is reminiscent of the agonist-induced internalization of N-type calcium channels observed in rat DRG neurons (Altier et al., 2006) and embryonic chick DRG neurons (Puckerin et al., 2006; Tombler et al., 2006). Prolonged exposure of opioid receptor-like receptor 1 (ORL1) receptors to the agonist nociceptin has been shown to trigger the internalization of the ORL1 receptors and Cav2.2 channels coexpressed in human embryonic kidney tsA-201 cells (Altier et al., 2006), which is accompanied by a tonic inhibition of calcium entry. Baclofen, a GABAB agonist, has also been shown to induce the rapid removal of ω-conotoxin GVIA-sensitive (N-type) calcium channels from the plasma membrane of DRG neurons (Tombler et al., 2006). A mechanism proposed for the internalization of voltage-dependent calcium channels involves the selective binding of β-arrestin 1 to the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-binding region of the calcium channel whereby, on GABAB receptor activation, receptors are recruited to the arrestin–channel complex and internalized (Puckerin et al., 2006). Furthermore, β-arrestins have been shown to be required to target nonreceptor tyrosine kinase c-Src to constitutively active G-protein-coupled μ-opioid receptors in DRG neurons, resulting in their internalization (Walwyn et al., 2007). It is likely, therefore, that α-conotoxin activation of GABAB receptors and the inhibition of N-type calcium channels in DRG neurons is mediated by a mechanism similar to that reported previously for internalization of GPCR–channel complexes.
In conclusion, our results identify Cav2.2 as a target of the analgesic α-conotoxins Vc1.1 and Rg1A. We propose a novel mechanism by which these α-conotoxins modulate N-type calcium channel currents in DRG neurons, that is, acting as agonists via G-protein-coupled GABAB receptors. The activation of GABAB receptors by agonists such as baclofen is well established to produce antinociceptive and antiallodynic actions in chronic pain models (Schuler et al., 2001; Bettler et al., 2004). Therefore, the activation of GABAB receptors by Vc1.1 and Rg1A and subsequent inhibition of Cav2.2 (N-type) channels is most likely the mechanism mediating their analgesic properties, despite their recently reported modulation, at lower efficacy, of the α9α10 nAChR (Vincler et al., 2006; Vincler and McIntosh, 2007).
This work was supported by an Australian Research Council (ARC) Discovery Grant (D.J.A., D.J.C.) and a National Health and Medical Research Council Program Grant (D.J.A.). G.B. is a University of Queensland Postdoctoral Research Fellow and D.J.C. is an ARC Professorial Fellow. We are grateful for helpful discussions with Prof. Mac Christie (University of Sydney, Sydney, Australia), Dr. Richard Lewis (University of Queensland, Brisbane, Australia), and Prof. Gerald Zamponi (University of Calgary, Calgary, Alberta, Canada).
- Correspondence should be addressed to Dr. David J. Adams, Queensland Brain Institute, The University of Queensland, Brisbane, QLD 4072, Australia.