Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the α1B subunit (Cav 2.2). The α1B-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to ω-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Lipid, sugar and liposaccharide based delivery systems 2

The vast majority of biologically active compounds will never be considered as potential drugs due to inherently poor bioavailability. This review discusses the progress in the development of chemical systems to improve the metabolic stability, absorption and physicochemical properties of potential drugs. Delivery systems that involve the conjugation of lipid and/or sugar moieties are highlighted, as well as novel methods of conjugation of these groups to drugs. The use of sugar molecules to target drugs to particular organs or cells is also discussed, as is the use of lipids in the growing area of gene delivery. This is an update of a previous review [1].

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs

Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. omega-Conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50-75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, omega-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 muM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 muM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 muM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons

1 The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2 Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. 3 Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. 4 Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced < 50% by CTX GVIA 10-100 nM. 5 Capsaicin (3 muM) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6 The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 muM) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 muM) reduced but did not abolish the remaining neurogenic dilations. 7 Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Cardiovascular actions of the venom from the Irukandji (Carukia barnesi) jellyfish: Effects in human, rat and guinea-pig tissues in vitro and in pigs in vivo

1. We have investigated the cardiovascular pharmacology of the crude venom extract (CVE) from the potentially lethal, very small carybdeid jellyfish Carukia barnesi, in rat, guinea-pig and human isolated tissues and anaesthetized piglets. 2. In rat and guinea-pig isolated right atria, CVE (0.1-10 mu g/mL) caused tachycardia in the presence of atropine (I mu mol/L), a response almost completely abolished by pretreatment with tetrodotoxin (TTX; 0.1 mu mol/L). In paced left atria from guinea-pig or rat, CVE (0.1-3 mu g/mL) caused a positive inotropic response in the presence of atropine (1 mu mol/L). 3. In rat mesenteric small arteries, CVE (0.1-30 mu g/mL) caused concentration-dependent contractions that were unaffected by 0.1 mu mol/L TTX, 0.3 mu mol/L prazosin or 0.1 mu mol/L co-conotoxin GVIA. 4. Neither the rat right atria tachycardic response nor the contraction of rat mesenteric arteries to CVE were affected by the presence of box jellyfish (Chironex fleckeri) antivenom (92.6 units/mL). 5. In human isolated driven right atrial trabeculae muscle strips, CVE (10 mu g/mL) tended to cause an initial fall, followed by a more sustained increase, in contractile force. In the presence of atropine (I mu mol/L), CVE only caused a positive inotropic response. In separate experiments in the, presence of propranolol (0.2 mu mol/L), the negative inotropic effect of CVE was enhanced, whereas the positive inotropic response was markedly decreased. 6. In anaesthetized piglets, CVE (67 mu g/kg, i.v.) caused sustained tachycardia and systemic and pulmonary hypertension. Venous blood samples demonstrated a marked elevation in circulating levels of noradrenaline and adrenaline. 7. We conclude that C. barnesi venom may contain a neural sodium channel activator (blocked by TTX) that, in isolated atrial tissue (and in vivo), causes the release of transmitter (and circulating) catecholamines. The venom may also contain a 'direct' vasoconstrictor component. These observations explain, at least in part, the clinical features of the potentially deadly Irukandji syndrome.

Conotoxins as research tools and drug leads

The complex mixture of biologically active peptides that constitute the venom of Conus species provides a rich source of ion channel neurotoxins. These peptides, commonly known as conotoxins, exhibit a high degree of selectivity and potency for different ion channels and their subtypes making them invaluable tools for unravelling the secrets of the nervous system. Furthermore, several conotoxin molecules have profound applications in drug discovery, with some examples currently undergoing clinical trials. Despite their relatively easy access by chemical synthesis, rapid access to libraries of conotoxin analogues for use in structure-activity relationship studies still poses a significant limitation. This is exacerbated in conotoxins containing multiple disulfide bonds, which often require synthetic strategies utilising several steps. This review will examine the structure and activity of some of the known classes of conotoxins and will highlight their potential as neuropharmacological tools and as drug leads. Some of the classical and more recent approaches to the chemical synthesis of conotoxins, particularly with respect to the controlled formation of disulfide bonds will be discussed in detail. Finally, some examples of structure-activity relationship studies will be discussed, as well as some novel approaches for designing conotoxin analogues.

Oxidative folding of the cystine knot motif in cyclotide proteins

The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Omega-conotoxins GVIA, MVIIA and CVID: SAR and clinical potential

Highly selective N-type voltage-gated calcium (Ca-V) channel inhibitors from cone snail venom (the omega-conotoxins) have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Earlier in 2005, Prialt ( Elan) or synthetic omega-conotoxin MVIIA, was the first omega-conotoxin to be approved by Food and Drug Administration for human use. This review compares the action of three omega-conotoxins, GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved N-type therapeutics that are more useful in the treatment of chronic pain.

Toxin insights into nicotinic acetylcholine receptors

Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. in the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin Iml. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR. (c) 2006 Elsevier Inc. All rights reserved.

Alpha-selenoconotoxins, a new class of potent alpha(7) neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one([ Sec(2,8)] ImI or [Sec(3,12)] ImI), or both([Sec(2,3,8,12)] ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha(7) nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.