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Neuronal (N)-type Ca2+channel-selective ω-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new ω-conotoxins, CVIE and CVIF, were discovered from a Conus catuscDNA library. Both conopeptides potently displaced 125I-GVIA binding to rat brain membranes. In Xenopus laevisoocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba2+currents through recombinant N-type (α1B-b/α2δ1/β3) Ca2+channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of ω-conotoxin action was investigated by creating molecular diversity in β subunits: N-type channels with β2asubunits almost completely recovered from CVIE or CVIF block, whereas those with β3subunits exhibited weak recovery, suggesting that reversibility of the ω-conotoxin block may depend on the type of β-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca2+channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that ω-conotoxin/voltage-gated Ca2+channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca2+channel-selective ω-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

The synthetic α-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the α9α10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14γ]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by α9α10 nAChRs. This suggests that α9α10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

The effects of verapamil and related phenylalkylamines on neuronal excitability were investigated in isolated neurons of rat intracardiac ganglia using whole-cell perforated patch-clamp recording. Verapamil (≥10 μM) inhibits tonic firing observed in response to depolarizing current pulses at 22°C. The inhibition of discharge activity is not due to block of voltage-dependent Ca2+channels because firing is not affected by 100 μM Cd2+. The K+channel inhibitors charybdotoxin (100 nM), 4-aminopyridine (0.5 mM), apamin (30–100 nM), and tetraethylammonium ions (1 mM) also have no effect on firing behavior at 22°C. Verapamil does not antagonize the acetylcholine-induced inhibition of the muscarine-sensitive K+current (M-current) in rat intracardiac neurons. Verapamil inhibits the delayed outwardly rectifying K+current with an IC50value of 11 μM, which is approximately 7-fold more potent than its inhibition of high voltage-activated Ca2+channel currents. These data suggest that verapamil inhibits tonic firing in rat intracardiac neurons primarily via inhibition of delayed outwardly rectifying K+current. Verapamil inhibition of action potential firing in intracardiac neurons may contribute, in part, to verapamil-induced tachycardia.

Brevetoxin-3 (PbTx-3), produced by marine dinoflagellates (Ptychodiscus brevis), is a lipophilic 11-ring polyether molecule that binds with high affinity to site 5 of the voltage-sensitive sodium (Na+) channel. The effects of PbTx-3 and its derivatives were studied in cell-attached membrane patches on neurons dissociated from neonatal rat nodose ganglia by the patch-clamp technique. PbTx-3 (30–500 nM) produced a shift in activation to more negative membrane potentials whereby single-channel activity was observed under steady-state conditions (maintained depolarization at −50 mV). The unitary current-voltage relationship is linear, which exhibits a reversal potential of approximately +60 mV. Two unitary current amplitudes could be observed in the presence of PbTx-3, with slope conductances of 10.7 pS and 21.2 pS. PbTx-3 inhibits the inactivation of Na+channels and prolongs the mean open time of these channels. Unitary Na+currents could be blocked by 1 μM tetrodotoxin (TTX) added to the pipette solution, which indicates that the single-channel currents are caused by the opening of TTX-sensitive Na+channels. The PbTx-3 molecule is proposed to have multiple active centers (A-ring lactone, C-42 of R side chain) interacting with the Na+channel binding site. Modification of the molecular structure of PbTx-3 at these centers produced derivatives (PbTx-6, 2,3,41,43-tetrahydro-PbTx-3, 2,3,27,28,41,43-hexahydro-PbTx-3 and 2,3-dihydro-PbTx-3 A-ring diol), which were less potent than PbTx-3 in producing similar effects on Na+channel kinetics. PbTx-3 and its derivatives may provide insight into the mechanics of voltage-sensitive Na+channel gating.