ω-Conotoxin GVIA

Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using omega-conotoxin GVIA and its selenocysteine analogs.[Pubmed:22782563]

Biopolymers. 2012;98(3):212-23.

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of omega-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.

Complex structures between the N-type calcium channel (CaV2.2) and omega-conotoxin GVIA predicted via molecular dynamics.[Pubmed:23651160]

Biochemistry. 2013 May 28;52(21):3765-72.

The N-type voltage-gated Ca(2+) channel CaV2.2 is one of the important targets for pain management. omega-Conotoxins isolated from venoms of cone snails, which specifically inhibit CaV2.2, are promising scaffolds for novel analgesics. The inhibitory action of omega-conotoxins on CaV2.2 has been examined experimentally, but the modes of binding of the toxins to this and other related subfamilies of Ca(2+) channels are not understood in detail. Here molecular dynamics simulations are used to construct models of omega-conotoxin GVIA in complex with a homology model of the pore domain of CaV2.2. Three different binding modes in which the side chain of Lys2, Arg17, or Lys24 from the toxin protrudes into the selectivity filter of CaV2.2 are considered. In all the modes, the toxin forms a salt bridge with an aspartate residue of subunit II just above the EEEE ring of the selectivity filter. Using the umbrella sampling technique and potential of mean force calculations, the half-maximal inhibitory concentration (IC50) values are calculated to be 1.5 and 0.7 nM for the modes in which Lys2 and Arg17 occlude the ion conduction pathway, respectively. Both IC50 values compare favorably with the values of 0.04-1.0 nM determined experimentally. The similar IC50 values calculated for the different binding modes demonstrate that GVIA can inhibit CaV2.2 with alternative binding modes. Such a multiple-binding mode mechanism may be common for omega-conotoxins.

Interference between two modulators of N-type (CaV2.2) calcium channel gating demonstrates that omega-conotoxin GVIA disrupts open state gating.[Pubmed:20471360]

Biochim Biophys Acta. 2010 Sep;1798(9):1821-8.

N-type calcium channels play an important role in synaptic transmission and a drug that blocks these channels has become an important tool in controlling chronic pain. The development of new N-channel-targeted drugs is dependent on a better understanding of the gating of these channels and how that gating can be modulated. We have previously concluded that omega-conotoxin GVIA (GVIA) is a gating modifier that acts by destabilizing the N-channel open state. However, this conclusion was largely based on our modeling results and requires experimental support. Roscovitine, a tri-substituted purine, has been shown to stabilize the N-channel open state to slow gating charge relaxation, which provides a direct test of our hypothesis for GVIA-induced gating modification. We found that roscovitine could modulate gating current in the presence of GVIA, which shows that roscovitine can still affect the gating of the GVIA-bound N-channel. However, the magnitude of the roscovitine-induced slowing of Off-gating current was significantly reduced. In addition to confirming our hypothesis, our evidence supports an additional effect of GVIA to alter gating transitions between N-channel closed states. By strongly limiting access to the N-channel open state, GVIA analogs that selectively induce this modulation could provide the basis for the next generation drugs that treat chronic pain.

omega-Conotoxin GVIA mimetics that bind and inhibit neuronal Ca(v)2.2 ion channels.[Pubmed:23170089]

Mar Drugs. 2012 Oct;10(10):2349-68.

The neuronal voltage-gated N-type calcium channel (Ca(v)2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived omega-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a (1)(2)(5)I-omega-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca(v)2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of omega-conotoxin GVIA and found to be active in the (1)(2)(5)I-omega-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.

Distinct mechanisms contribute to agonist and synaptically induced metabotropic glutamate receptor long-term depression.[Pubmed:21575629]

Eur J Pharmacol. 2011 Sep 30;667(1-3):160-8.

Metabotropic glutamate receptor mediated long-term depression (mGlu receptor LTD) is evoked in hippocampal area CA1 chemically by the agonist 3,5-Dihydroxyphenylglycine (DHPG, agonist LTD) and synaptically by paired-pulse low frequency stimulation (PP-LFS, synaptic LTD). We tested the hypothesis that different expression mechanisms regulate mGlu receptor LTD in 15-21 day old rats through neurophysiologic recordings in CA1. Several findings, in fact, suggest that agonist and synaptic mGlu receptor LTD are expressed through different mechanisms. First, neither LTD occluded the other. Second, a low calcium solution enhanced agonist LTD but did not alter synaptic LTD. Third, application of the mGlu receptor antagonist LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid) reversed agonist LTD expression, but did not alter synaptic LTD. Finally, an N-type, voltage-dependent calcium channel antagonist, omega-conotoxin GVIA (CTX), reduced agonist LTD expression by 35%, but did not alter synaptic LTD. CTX also blocked the increase in the paired-pulse ratio associated with agonist LTD. This study cautions against assuming that agonist and synaptic LTD are equivalent as several components underlying their expression appear to differ. Moreover, the data suggest that agonist LTD, but not synaptic LTD, has a presynaptic, N-channel mediated component.

Prolonged cardiovascular effects of the N-type Ca2+ channel antagonist omega-conotoxin GVIA in conscious rabbits.[Pubmed:9300325]

J Cardiovasc Pharmacol. 1997 Sep;30(3):392-9.

omega-Conotoxin GVIA (omega-CTX) is an N-type Ca2+ channel antagonist that is considered to be only partially reversible in vitro. In vivo, its effects after 24 h are unknown. To assess the duration of action of this peptide in vivo, the effects of a single intravenous injection of omega-CTX on mean arterial pressure (MAP), heart rate (HR), postural adaptation, and the baroreflex were investigated in conscious rabbits. MAP, HR, the baroreflex induced by i.v. glyceryl trinitrate (0.4-20 micrograms/kg) and phenylephrine (0.1-15 micrograms/kg) and orthostatic responses to 1 min 90 degrees head-up tilt were assessed before (0 h) and 2-168 h after administration of omega-CTX (10 micrograms/kg i.v. bolus: n = 6-9) or vehicle (0.9% saline; n = 6). Acute phase I: By 2 h after omega-CTX administration, MAP had decreased from 75 +/- 3 mm Hg to 60 +/- 2 mm Hg; HR increased from 220 +/- 7 beats/min to 249 +/- 5 beats/min (n = 9). There was marked attenuation of the baroreflex curve (HR range decreasing by 61%). By 24 h. MAP and HR had returned to control values, but the HR range was still 18% less than that of control. Phase II: MAP and HR then decreased steadily over the next 96 h to significantly lower values by 120 h after omega-CTX administration (delta-8 +/- 2 mm Hg and -29 +/- 2 beats/min, respectively; n = 6). Thereafter, MAP and HR values increased and by 168 h these parameters, and the baroreflex, were similar to control values. In response to 90 degrees tilt, there was no change in MAP at 0 h; however, 1 h after omega-CTX, significant postural hypotension was observed with decreases of 14 +/- 1 mm Hg(n = 9). Smaller orthostatic responses were still observed 48 h after omega-CTX administration: however, by 72 h, head-up tilt no longer induced a significant change in MAP. In the vehicle-treatment group, there were no changes in cardiovascular parameters during 0-168 h. Thus omega-CTX (10 micrograms/kg i.v.) causes acute hypotension, as well as postural hypotension, and has sympatholytic and vagolytic effects that are mostly reversed after 48 h in the conscious rabbit. However, a second hypotensive and bradycardic phase lasting a further 96 h ensues, suggesting that other prolonged effects from central neural or hormonal mechanisms or fluid shifts may occur.

Role of basic residues for the binding of omega-conotoxin GVIA to N-type calcium channels.[Pubmed:8394704]

Biochem Biophys Res Commun. 1993 Aug 16;194(3):1292-6.

Each of four basic residues of omega-conotoxin GVIA was replaced with alanine to study the role of basic residues for the binding of this toxin to N-type calcium channels. The activities of these analogs were estimated from the inhibitory action on 125I-omega-conotoxin GVIA binding to chick brain synaptic plasma membranes. The replacement of Arg17, Lys24 and Arg25 resulted in no significant change in the activity and all of the analogs gave the same IC50 value (0.15 nM) as that of native omega-conotoxin GVIA. The inhibitory action of [Ala2]omega-conotoxin GVIA (K2A) was 40-times less potent (IC50 = 5.5 nM); however, full inhibition was achieved at a concentration above 0.1 microM. These results indicate that the Arg residue is not essential for the activity of omega-conotoxin GVIA. The nature of association to ion channels may be different between omega-conotoxin GVIA and mu-conotoxin GIIIA.