Co-Conotoxin GVIA

Air oxidation of synthetic fully reduced co-conotoxin GVIA (9) (Scheme 4), a 27-residue peptide, 64 generated a main product that possessed the identical biological activities as the natural product whose cystine framework was determined with the synthetic compound. 65 The choice of buffer system and peptide concentration can, however, dramatically affect the folding efficiency when the DMSO-mediated oxidation method was applied. 661... 

Results similar to those reported for co-conotoxin GVIA were obtained with the Na+, K+-ATPase inhibitor-I (10) J67 a 49 amino acid residue peptide containing four intramolecular disulfide bonds (Scheme 5)J681 Even in this case the addition of redox reagents to the oxidation buffer had a minor effect on the product distribution. 

All the disulfide-folding pathways have the common feature that disulfide formation is initially random and not energetically favored due to the unfolded state of the reduced polypeptide chain. Disulfide formation becomes less random as certain disulfides are favored energetically as a result of the acquisition of nonrandom local conformations. In turn, the disulfides stabilize such conformations, so that disulfide formation and folding become cooperative. Thereby the sequence encoded structural information may fully suffice to direct the correct oxidative refolding as shown for co-conotoxin GVIA (9) (Section 6.1.5.2.1) which assumes the native-type cystine connectivities directly even in absence of redox reagents and... 

Unlike DHP-sensitive L-type channels co-conotoxin-sensitive N-type Ca2+ channels are exclusively expressed in the CNS. L-type channels inactivate very slowly whereas N-type channels inactivate more rapidly and are blocked by co-conotoxin GVIA. co-[125l]conotoxin GVIA is an ideal ligand for binding experiments. The dissociation constant (KD) for this toxin in rat brain membranes is 60 pM (Wagner et al., 1988). 

P-type channels inactivate extremely slowly and are insensitive to both DHPs and co-conotoxin GVIA, but are blocked by the spider venom peptide co-agatoxin IVA, a peptide consisting of 48 amino acids isolated from the American funnel-web spider Agelenopsis aperta. 

The inactivation kinetics of the Q-type channel are similar to the N-type channel but are resistant to DHPs and co-conotoxin GVIA. Q-type channels are inhibited by co-agatoxin IVA but less effectively than co-agatoxin IVA blocks P-type channels (review Miljanich and Ramachandran, 1995). 

Wagner,J.A., Snowman, AM., Biswas, A., Olivera, B.M., Snyder, S.H. co-Conotoxin GVIA binding to a high-affinity receptor in brain Characterization, calcium sensitivity, and solubilization, J. Neurosci. 1988, 8, 3354- 3359. 

Cav2.2 N-type CtlB presynaptic nerve terminals evoked neurotransmitter release co-conotoxin GVIA, MVIIA and CVID, (D-grammotoxin SIVA, farnesol, peptidylamines hyponociceptive, reduced anxiety, reduced withdrawal symptoms... 

Pharmacologically, N-type channels are characterized by the irreversible blockade induced by the C. geographus toxin co-conotoxin GVIA [2,46,67] and the reversible blockade induced by the... 

N-channel currents have been characterized in chromaffin cells of various species including bovine [57,69], pig [63], cat[64],rat[47],mouse[62],andhuman [65]. This current suffers voltage-dependent inactivation [34,70], but see reference [57], and is irreversibly blocked by co-conotoxin GVIA [11] and co-conotoxin MVllC [16,17] or reversibly blocked by co-conotoxin MVllD... 

P-type Ca channels were first described by Llinas et al. [29] in cerebellar Purkinje cells, in which Ca currents were resistant to blockade by DHPs and co-conotoxin GVIA. The toxin fraction from the venom of the funnel web spider A. aperta (FTX) was found effectively to block this resistant current, and these results led these authors to suggest the existence of a new subtype of HVA Ca " channel, which was termed P (for Purkinje ). 

Nanomolar concentrations of co-agatoxin IVA known to fully and selectively block P-type channels [22,72], cause only a 5-10% blockade of Ca channel current in bovine chromaffin cells [79]. Previous studies reported larger contributions of P-type channels to the whole chromaffin cell Ca " currents however, this blockade is now attributable to inhibition of Q-type channels [80] by sFTX [81 ] or large concentrations of co-agatoxin IVA [55,82]. In cat chromaffin cells, combined co-conotoxin GVIA plus nisoldipine blocked 90% of the current, leaving little room for P-type channels [64], In rat [47] and mouse chromaffin cells [62], the co-agatoxin IVA-sensitive current fraction was only... 

In many neuronal preparations, a significant component of the whole-cell current through Ca channels is resistant to blockade with DHPs, co-conotoxin GVIA, and co-agatoxin IVA (<100 nM), suggesting the presence of a subtype of Ca channel different from L-, N-, and P-types. The isolation, purification, and synthesis of the toxin from the marine snail C. magus co-conotoxin MVllC [16,68] led to the identification and characterization of a new subtype of HVA channel termed Q [80,84]. 

Characterization of Q-type Ca channels is mostly based on pharmacological criteria. As described, Q-type channels are resistant to blockade by DHPs, co-conotoxin GVIA, and low doses (<100 nM) of co-agatoxin IVA, but they are sensitive to co-conotoxin MVIIC (1-3 pM). Increasing concentrations of co-agatoxin IVA (up to 2 pM) can also block Q-type Ca channels [80]. It should... 

In neuronal tissues, a residual Ca current, characterized by its insensitivity to blockade by DHPs, co-conotoxin GVIA, co-agatoxin IVA, and co-conotoxin MVIIC has also been described and termed R-type (for resistant ) [84], This new subtype of Ca channel belongs to the HVA group, is rapidly inachvating (x = 22 ms), and more sensitive to blockade by Ni (IC50 = 66 pM) than to Cd ". ... 

Glutamatergic synaptic responses in rat retinal ganglion neurons are partially sensitive to co-conotoxin GVIA (30% block) and insensitive to co-agatoxin IVA [151], These results indicate that the major part of synaptic glutamate release in retinal ganglion neurons is governed by a novel toxin-resistant Ca " channel that could possibly be of the Q or R type. 

In cocultures of fetal neurons from ventral half of the spinal cord (VH neurons) and from the dorsal root ganglion (DRG neurons) the synaptic transmission between pairs of spinal cord neurons from ventral half of the spinal cord (VH-VH connections) or between DRG neurons and VH neurons (DGR and VH connections) were studied with two cell recording and stimulation techniques. In 70% of the VH-VH connections and in 50% of the DGR-VH connections, Bay K 8644 failed to affect transmitter release. co-Conotoxin GVIA produced no consistent effect on EPSPs or IPSPs elicited by VH neurons by stimulation of the nearby neurons. VH EPSPs elicited by stimulation of the nearby DGR neurons were reduced by 50% by co-couotoxiu GVIA. Therefore, ueither sustaiued nor inactivating HVA Ca channels sensitive to Bay K 8644 or co-conotoxin GVIA such as those measured in the neuronal cell body are responsible for action-potential-evoked transmitter release from the majority of the VH neurons these channels may be involved in transmitter release in approximately 30% of these neurons [152]. 

In rat dorsal horn neurons of the spinal cord, release of glycine induces IPSCs. The IPSCs were almost completely blocked by co-conotoxin GVIA (more than 95%) and partially inhibited by co-agatoxin IVA (50%), while nicardipine had no effect. 

Electrically evoked release of acetylcholine is predominantly controlled through N-type Ca channels at the myenteric plexus [98,153,154]. co-Conotoxin GVIA markedly reduced (70%) the evoked release of [ H]-acetylcholine from the myenteric plexus of the small intestine, with an IC50 of... 

In the guinea-pig jejunum, co-conotoxin GVIA blocked only partially (33%) the inhibitory NANC transmission upon electrical stimulation. This was also the case at the taenia caecum (20% inhibition) [154]. In the proximal duodenum the NANC transmission was insensitive to co-conotoxin GVIA [155]. 

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