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At least 19 recordsLinked to original sources
PubMed1994

Omega-agatoxin-TK containing D-serine at position 46, but not synthetic omega-[L-Ser46]agatoxin-TK, exerts blockade of P-type calcium channels in cerebellar Purkinje neurons.

omega-Agatoxin-TK (omega-Aga-TK), a 48-amino-acid peptide isolated from the venom of the funnel web spider (Agelenopsis aperta), is a selective and potent inhibitor of P-type calcium channels in the nervous system. We have synthesized a peptide that has the amino acid sequence identified for native omega-Aga-TK. The synthetic omega-Aga-TK, however, showed 80-90-fold less potent inhibition of P-type calcium channels, compared with native omega-Aga-TK. Enantiomer analysis of native omega-Aga-TK revealed D-serine at position 46, and synthetic omega-[D-Ser46]Aga-TK had the same potency as native omega-Aga-TK for blocking P-type calcium channels in cultured cerebellar Purkinje neurons. Two peptide fragments of omega-Aga-TK, namely omega-Aga-TK(1-43) and the carboxyl-terminal peptide fragment omega-Aga-TK(44-48), did not produce any significant inhibition of P-type calcium channels or interfere with the blockade of the channels elicited by native omega-Aga-TK. Molecular dynamics calculations showed that the carboxyl-terminal, six-amino-acid peptide of omega-Aga-TK containing D-Ser46 assumes a different conformation than does the peptide containing L-Ser46. These results suggest that the specific conformation of the carboxyl-terminal region of omega-Aga-TK, particularly the configuration of Ser46, together with a beta-sheet structure formed by four disulfide bonds, might be essential for blockade of P-type calcium channels.

Agatoxins↗
PubMed2005

Omega-agatoxin-TK is a useful tool to study P-type Ca2+ channel-mediated changes in internal Ca2+ and glutamate release in depolarised brain nerve terminals.

The present study shows that omega-agatoxin-TK, a toxin of the venom of Agelenopsis aperta, which is 10 times more concentrated than the P/Q type Ca(2+) channel blocker, omega-agatoxin-IVA in the venom, inhibits the high K(+) depolarisation-induced rise in internal Ca(2+) (Ca(i), as determined with fura-2) dose dependently in cerebral (striatal and hippocampal) isolated nerve endings, with calculated IC(50)'s of about 60nM. The maximal inhibition exerted by omega-agatoxin-TK in striatal synaptosomes (61 +/- 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of omega-agatoxin-TK-sensitive Ca(2+) channels in striatal than in hippocampal nerve endings. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1muM), inhibits part of the omega-agatoxin-TK-insensitive rise in Ca(i) induced by high K(+). In contrast to the inhibition exerted by omega-agatoxin-TK on the Ca(i) response to high K(+), omega-agatoxin-TK failed to inhibit the tetrodotoxin-sensitive elevations in Ca(i) and in internal Na(+) (Na(i), as determined with SBFI) induced by veratridine, indicating that the Ca(2+) influx activated by veratridine does not involve omega-agatoxin-TK-sensitive channels. High K(+) does not increase Na(i). In [(3)H]Glu preloaded hippocampal synaptosomes super-fused with low Na(+) Krebs Ringer HEPES (a condition that guarantees the elimination of neurotransmitter transporters-mediated release), the release of [(3)H]Glu induced by high K(+) is absolutely dependent on the entrance of external Ca(2+). This exocytotic release of [(3)H]Glu attained in the absence of a chemical Na(+) gradient is inhibited with the same potency and efficacy by omega-agatoxin-TK and by omega-agatoxin-IVA, which is known to differ from omega-agatoxin-TK in its amino terminal moiety. These results indicate that omega-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca(2+) channel-mediated responses in cerebral nerve endings.

Agatoxins↗
PubMed1992

Omega-agatoxins differentially block calcium channels in locust, chick and rat synaptosomes.

Three toxins (omega-Agatoxins IA, IIA and IIIA) isolated from the venom of the funnel web spider, Agelenopsis aperta, differentially block depolarization-induced calcium influx in chick, rat and locust synaptosomes. In chick, this block of calcium influx is observed with omega-Agatoxins IIA and IIIA but not with omega-Agatoxin IA. Block by omega-Agatoxin IIA and IIIA is maximal at 70 and 82% respectively of the total depolarization-induced calcium influx; maximal suppression of calcium influx by omega-Conotoxin GVIA (omega-CgTx) is 100%. The IC50 for block with omega-Agatoxin IIA is ca 3 nM as compared with an IC50 of 38 nM for omega-CgTx. Incomplete block of calcium influx at saturating concentrations of omega-Agatoxins IIA and IIIA (above 100 nM) suggests that both omega-Agatoxin-sensitive and -insensitive calcium channels occur in chick brain synaptosomes. In rat cerebrocortical synaptosomes, omega-Agatoxins IA and IIA are only partially effective at blocking depolarization-induced calcium influx, as is omega-CgTx, whilst IIIA blocks 47% of this effective at blocking depolarization-induced calcium influx, as is omega-CgTx, whilst IIIA blocks 47% of this influx. In synaptosomes prepared from the CNS of adult locusts, omega-Agatoxins IA and IIA are most effective at blocking depolarization-induced calcium influx; omega-CgTx and omega-Agatoxin IIIA are ineffective. Block of depolarization-induced calcium influx in chick brain synaptosomes by omega-Agatoxins IIA, IIIA and omega-CgTx suggests that the spider toxin interacts directly with the voltage-dependent calcium channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Agatoxins↗
PubMed2004

Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta.

Agatoxins from Agelenopsis aperta venom target three classes of ion channels, including transmitter-activated cation channels, voltage-activated sodium channels, and voltage-activated calcium channels. The alpha-agatoxins are non-competitive, use-dependent antagonists of glutamate receptor channels, and produce rapid but reversible paralysis in insect prey. Their actions are facilitated by the micro-agatoxins, which shift voltage-dependent activation of neuronal sodium channels to more negative potentials, causing spontaneous transmitter release and repetitive action potentials. The omega-agatoxins target neuronal calcium channels, modifying their properties in distinct ways, either through gating modification (omega-Aga-IVA) or by reduction of unitary current (omega-Aga-IIIA). The alpha-agatoxins and omega-agatoxins modify both insect and vertebrate ion channels, while the micro-agatoxins are selective for insect channels. Agatoxins have been used as selective pharmacological probes for characterization of ion channels in the brain and heart, and have been evaluated as candidate biopesticides.

Amino Acid Sequence↗
PubMed1996

Dihydropyridine block of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat pituitary melanotropic cells.

High voltage-activated Ca2+ currents in rat melanotropic cells consist of a sustained and an inactivating component. In this study the pharmacological properties of the high voltage-activated Ca2+ channels underlying these components are investigated with whole-cell recordings. We report that melanotropes express four pharmacologically distinct high voltage-activated Ca2+ channels. Non-inactivating L-type channels account for 35% of the total high voltage-activated channel population. These channels have a very high affinity for the dihydropyridine nimodipine (EC50 approximately 3 pM). The cone snail toxin omega-conotoxin GVIA irreversibly blocked an inactivating high voltage-activated component which accounted for 26% of the total whole-cell high voltage-activated Ca2+ current. The spider toxin omega-agatoxin IVA reversibly blocked an additional 31% of the total high voltage-activated current. The current blocked by omega-agatoxin IVA was not homogenous and consisted of a sustained component with a high affinity for omega-agatoxin IVA (< 10 nM) and an inactivating current with a low affinity for omega-agatoxin IVA (> 100 nM). Both the omega-agatoxin IVA and omega-conotoxin GVIA-blocked currents were very sensitive to nimodipine and nitrendipine with a half maximal block at 200-500 nM. 10 microM nimodipine blocked 70% of the omega-conotoxin GVIA-sensitive current and 90% of the omega-agatoxin IVA-sensitive current. Thus, omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive high voltage-activated Ca2+ channels in melanotropes have an unusual high affinity for dihydropyridines compared to N-, P-, and Q-type channels in other preparations.

Animals↗
PubMed1991

Differential antagonism of transmitter release by subtypes of omega-agatoxins.

1. The omega-agatoxins from Agelenopsis aperta spider venom are a diverse group of voltage-sensitive calcium channel antagonists. Subtypes of omega-agatoxins are distinguished as type I (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IC), type II (omega-Aga-IIA and omega-Aga-IIB), and type III (omega-Aga-IIIA and omega-Aga-IIIB). All except type III toxins block calcium channels in insect motor nerve terminals and in neuronal cell bodies at nanomolar concentrations. 2. The potency and maximum level of block of the excitatory junctional potential (EJP) by omega-agatoxins are dependent on the extracellular calcium concentration ([Ca]o). Saturating concentrations of type I or II omega-agatoxins block 97-99% of the evoked EJP in low [Ca]o (0.75 mM) saline. A remnant of the EJP (1-3%) that persists after toxin exposure suggests that a small amount of voltage-dependent calcium entry is toxin resistant. When [Ca]o is elevated to 5 mM, this resistant component increases dramatically, revealing differences between type I and type II block. Under these conditions, 60-70% of the EJP is resistant to type I toxins and approximately 20% is resistant to type II toxins. 3. Sequential application of type I and II toxins in high [Ca]o leads to enhanced block of the EJP, suggesting that type I and II omega-agatoxins may block calcium channels by different mechanisms. 4. Type I and type II omega-agatoxins also block calcium channels in the somata of locust dorsal unpaired median (DUM) neurons. In agreement with studies on neuromuscular transmission, block of barium action potentials is incomplete after either type I or type II toxin exposure and combined application of the toxins results in enhanced block. 5. Partial calcium channel antagonism by type I and type II toxins could be explained either by altered kinetics of toxin-modified channels or by selectivity for different subtypes of presynaptic calcium channels.

Agatoxins↗
PubMed1999

Effects of omega-agatoxin IVA, a P-type calcium channel antagonist, on the development of spinal neuronal hyperexcitability caused by knee inflammation in rats.

Both N- and P-type high-threshold calcium channels are located presynaptically in the CNS and are involved in the release of transmitters. To investigate the importance of P-type calcium channels in the generation of inflammation-evoked hyperexcitability of spinal cord neurons, electrophysiological recordings were made from wide-dynamic-range neurons with input from the knee joint in the anesthetized rat. The responses of each neuron to innocuous and noxious pressure onto the knee and the ankle were continuously assessed before and during the development of an inflammation in the knee joint induced by the injections of K/C into the joint cavity. The specific antagonist at P-type calcium channels omega-agatoxin was administered into a 30-microl trough on the spinal cord surface above the recorded neuron. In most neurons the application of omega-agatoxin before induction of inflammation slightly enhanced the responses to pressure onto the knee and ankle or left them unchanged. Two different protocols were then followed. In the control group (13 rats) only Tyrode was administered to the spinal cord during and after induction of inflammation. In these neurons the responses to mechanical stimuli applied to both the inflamed knee and to the noninflamed ankle showed a significant increase over 4 h. In the experimental group (12 rats) omega-agatoxin was applied during knee injection and in five 15-min periods up to 180 min after kaolin. This prevented the increase of the neuronal responses to innocuous pressure onto the knee and to innocuous and noxious pressure onto the ankle; only the responses to noxious pressure onto the knee were significantly enhanced during development of inflammation. Thus the development of inflammation-evoked hyperexcitability was attenuated by omega-agatoxin, and this suggests that P-type calcium channels in the spinal cord are involved in the generation of inflammation-evoked hyperexcitability of spinal cord neurons. Finally, when omega-agatoxin was administered to the spinal cord 4 h after the kaolin injection, i.e., when inflammation-evoked hyperexcitability was fully established, the responses to innocuous and noxious pressure onto the knee were reduced by 20-30% on average. The shift in the effect of omega-agatoxin, from slight facilitation or no change of the responses before inflammation to inhibition in the state of hyperexcitability, indicates that P-type calcium channels are important for excitatory synaptic transmission involved in the maintenance of inflammation-evoked hyperexcitability.

Animals↗
PubMed1995

Spatial distribution of omega-agatoxin IVA binding sites in mouse brain slices.

A peptide toxin derived from funnel-web spider venom, omega-agatoxin IVA, blocks voltage-sensitive calcium channels. Many pharmacological and electrophysiological studies have shown that these channels are widely distributed in both the central nervous system (CNS) and neuromuscular junctions. However, a direct morphological demonstration of the binding sites of this toxin is still lacking. To identify which cells have the binding sites, a biologically active, biotin-conjugated omega-agatoxin IVA was applied to mouse cerebellar and hippocampal slices. Confocal microscopy revealed that omega-agatoxin IVA binding sites were distributed on the somata of Purkinje cells, cerebellar granule cells and interneurons, as well as on the dendrites of Purkinje cells. In the hippocampus, the binding sites were localized on the somata of pyramidal cells of the CA1-CA4 region and on the somata of granule cells in the dentate gyrus. A sequential competitive reaction confirmed the specificity of the binding in the cerebellum and CA1 pyramidal cells, and also suggested a difference in the binding affinity between CA1 and CA3 pyramidal cells. Since a high concentration of omega-agatoxin IVA (2 microM) was needed for the present study, the omega-agatoxin IVA binding sites presented in this study may represent "P-type" and "Q-type" calcium channels.

Animals↗
PubMed1995

Inhibition of acetylcholine release from mouse motor nerve by a P-type calcium channel blocker, omega-agatoxin IVA.

1. The effects were studied of the central neurone P-type Ca2+ channel blockers, omega-agatoxin IVA, omega-conotoxin MVIIC (polypeptide toxins) and synthetic funnel-web spider polyamine toxin on acetylcholine release from mouse motor nerve. 2. omega-Agatoxin IVA decreased the quantal content of endplate potentials and blocked synaptic transmission in the nanomolar range in a reversible manner, whereas the other toxins depressed transmission in the hundred micromolar range. 3. The polyamine toxin, but not the polypeptide toxins, decreased the amplitude of the miniature endplate potential. The increase in the frequency of miniature endplate potentials evoked by high [K+], but not that evoked by alpha-latrotoxin, was effectively antagonized by omega-agatoxin IVA. 4. In the presence of omega-agatoxin IVA, high frequency nerve stimulation produced facilitation of endplate currents and tetanic contractions. 5. The results suggest that, under physiological conditions, the Ca2+ necessary for nerve action potential-evoked acetylcholine release is translocated via a subtype of the P-type Ca2+ channel sensitive to omega-agatoxin IVA.

Acetylcholine↗
PubMed1995

Three-dimensional solution structure of the calcium channel antagonist omega-agatoxin IVA: consensus molecular folding of calcium channel blockers.

The three-dimensional solution structure of omega-agatoxin IVA, which is a specific blocker of the P-type calcium channel isolated from funnel web spider venom and has a molecular mass of 5.2 kDa, was determined by two dimensional 1H NMR spectroscopy, combined with simulated annealing calculations. On the basis of 563 experimental constraints, including 516 distance constraints obtained from the nuclear Overhauser effect, 21 torsion angle (phi, chi 1) constraints, and 26 constraints associated with hydrogen bonds and disulfide bonds, a total of 14 converged structures were obtained. The atomic root mean square difference for the 14 converged structures with respect to the mean coordinates is 0.42 (+/- 0.07) A for the backbone atoms (N, C alpha, C) and 0.95 (+/- 0.15) A for all heavy atoms of the central part (residues 4 to 38) constrained by four disulfide bonds. The N- and C-terminal segments (residues 1 to 3 and 39 to 48, respectively) have a disordered structure in aqueous solution. The molecular structure of omega-agatoxin IVA is composed of a short triple-stranded antiparallel beta-sheet, three loops, and the disordered N- and C-terminal segments. The overall beta-sheet topology is +2x, -1, which is the same as that reported for omega-conotoxin GVIA, an N-type calcium channel blocker. Irrespective of differences in the number of disulfide bonds and low primary sequence homology, these two peptide toxins show a significant structural similarity in three dimensions. The whole-cell voltage-clamp recording using rat cerebellar slices suggests that the hydrophobic C-terminal segment of omega-agatoxin IVA, which does not exist in omega-conotoxin GVIA, plays a crucial role in the blocking action of omega-agatoxin IVA on the P-type calcium channel in rat cerebellar Purkinje cells. The present study provides a molecular basis for the toxin-channel interaction, and thereby provides insight into the discrimination of different subtypes of calcium channels.

Amino Acid Sequence↗
PubMed1997

Two types of omega-agatoxin IVA-sensitive Ca2+ channels are coupled to adrenaline and noradrenaline release in bovine adrenal chromaffin cells.

To clarify the role of P-type Ca2+ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of omega-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K(+)-evoked depolarization. omega-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component (IC50 < 1 nM), responsible for 10-15% of catecholamine release evoked by 70 mM K+, and a low-potency component that accounted for about 40% of release, with IC50 values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin cells was also inhibited dose dependently by omega-conotoxin MVIIC with IC50 values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM omega-agatoxin IVA and 3 microM omega-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites, presumably, P- and Q-type Ca2+ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 +/- 4.1%) significantly more than the release of noradrenaline (50 +/- 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of K(+)-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show that two types of omega-agatoxin IVA-sensitive Ca2+ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion is larger at low levels of depolarization.

Adrenal Glands↗
PubMed1994

Effect of omega-agatoxin-IVA on autonomic neurotransmission.

omega-Agatoxin-IVA, a peptide from the venom of the funnel-web spider Agelenopsis aperta and a P type Ca2+ channel inhibitor, was examined for effects on responses to nerve stimulation in isolated autonomic neuroeffector preparations from the rabbit, guinea-pig and rat. Ca(2+)-dependent, tetrodotoxin sensitive, noradrenergic excitatory responses of rabbit pulmonary artery, rat vas deferens, and anococcygeus muscles, and cholinergic guinea-pig myenteric plexus preparations (all highly sensitive to the N type Ca2+ channel inhibitor omega-conotoxin-GVIA) were unaffected by omega-agatoxin-IVA (100 nM). Similarly, the neurogenic response of rat bladder, which has cholinergic, and non-adrenergic non-cholinergic (NANC) excitatory components, and the NANC inhibitory response of rat jejunum (atropine 0.5 microM- and guanethidine 5.0 microM-treated), which are partially sensitive and insensitive to omega-conotoxin-GVIA, respectively, were unaffected by omega-agatoxin-IVA (100 nM). Neurogenic NANC inhibitory responses of the guinea-pig taenia caecum, and rat anococcygeus muscles (atropine- and guanethidine-treated, and tone raised with prostaglandin F2 alpha), were also insensitive to omega-agatoxin-IVA. These results suggest that P type Ca2+ channels, if present, play an insignificant role in supplying the Ca2+ necessary for neurotransmitter release in the peripheral autonomic nervous system.

Animals↗
PubMed1995

omega-Agatoxin IVA blocks nicotinic receptor channels in bovine chromaffin cells.

We have studied the contribution of P-type voltage-dependent Ca2+ channels to both catacholamine (CA) and ATP secretion from bovine chromaffin cells induced by high K+ or nicotine using omega-agatoxin IVA, a selective blocker of P-type voltage-dependent Ca2+ channels. We found that high K+ (75 mM) induced the release of about 13% of norepinephrine, 5% epinephrine and 11% ATP, and that omega-agatoxin (100 nM) did not affect this secretion. However, both nicotine-induced CA and ATP secretion were significantly blocked (about 50%) by omega-agatoxin IVA (100 nM). In addition, this toxin also reversibly blocked (about 70%) the inward current induced by nicotine in bovine chromaffin cells. The results suggest that, besides its known action of blocking P-type voltage-dependent channels, omega-agatoxin is a potent and reversible blocker of the nicotinic receptor channel in chromaffin cells, and that this action would explain the blockade of nicotine-induced secretion.

Adenosine Triphosphate↗
PubMed1996

omega-agatoxin IVA and excitotoxicity in cortical neuronal cultures.

Because the P- and Q-type Ca2+ channel antagonist omega-agatoxin IVA inhibits glutamate release, agatoxin-related drugs could have a role in the treatment of excitotoxic neurologic disorders such as stroke. We examined the effect of omega-agatoxin IVA on excitotoxicity (lactate dehydrogenase release) induced by depolarization with veratridine or ouabain, or by N-methyl-D-aspartate, in neuron-enriched cerebral cortical cultures. omega-Agatoxin IVA (< or = 300 nM) failed to reduce excitotoxicity, as did the L-type Ca2+ channel antagonist nimodipine and the N-type antagonist omega-conotoxin GVIA. Drugs that inhibit glutamate release may not necessarily be neuroprotective.

Animals↗
PubMed1995

Physiological and regenerative acetylcholine release from motor nerve: differential inhibitions by vesamicol and omega-agatoxin IVA.

Stimulation of mammalian motor neurons can elicit Ca(2+)-dependent regenerative release of acetylcholine and prolonged endplate depolarization when the enzymatic degradation of the neurotransmitter is inhibited. Unlike physiological phasic release of acetylcholine, the regenerative release is sensitive to L-type Ca2+ channel blockers. We studied the effects of vesamicol (an inhibitor of active transport of acetylcholine into synaptic vesicles) and omega-agatoxin IVA (a blocker of the motor nerve P-type Ca2+ channel) on these two types of acetylcholine release to compare the vesicle pools and Ca2+ channels responsible for the release. When coupled with repetitive stimulations, vesamicol decreased mean amplitude of miniature endplate potentials, resulting in a skewed distribution to lower amplitude, reduced quantal content of endplate potentials and decreased immediate available pool of acetylcholine. omega-Agatoxin IVA had no effect on miniature endplate potential but inhibited quantal content of endplate potential. The mean inhibitory concentration was around 5-10 nM. Vesamicol and omega-agatoxin IVA decreased the probability of triggering regenerative release. However, the magnitude and duration of regenerative release, once triggered, were not depressed by either agent. It appears that the majority of Ca2+ necessary for regenerative release is translocated via omega-agatoxin IVA-insensitive Ca2+ channels, which can be activated by prolonged depolarization of nerve terminals induced by accumulated acetylcholine. The results suggest that different Ca2+ channels are activated in the regenerative (L-type) and phasic (P-type) acetylcholine release, which utilize different pools of synaptic vesicles.

Acetylcholine↗
PubMed1996

Omega-agatoxin IVA blocks spinal morphine/clonidine antinociceptive synergism.

Involvement of P-type voltage-dependent Ca2+ channels in spinal morphine- or clonidine-induced antinociception and in the synergistic interaction between morphine and clonidine was examined in the present studies. Coadministration of the selective P-type antagonist, omega-agatoxin IVA (25 ng) intrathecally (i.t.) to mice along with morphine or clonidine enhanced the tail flick antinociception of each agonist 5-6-fold. The greater-than-additive (synergistic) interaction that occurred when morphine and clonidine were coadministered i.t. decreased to an additive interaction in the presence of omega-agatoxin IVA. In mice pretreated with pertussis toxin (10 ng) to inactivate G proteins, omega-agatoxin IVA did not alter the morphine/clonidine synergism. Surprisingly, omega-agatoxin IVA reversed the additive morphine/clonidine interaction that occurs in morphine-tolerant mice back to synergism. These results suggest that functional P-type Ca2+ channels play an essential role in the antinociceptive synergism between spinal morphine and clonidine.

Analgesics, Opioid↗
PubMed1996

Three-dimensional structure analysis of mu-agatoxins: further evidence for common motifs among neurotoxins with diverse ion channel specificities.

We report the solution structure of mu-agatoxin-I (mu-Aga-I) and model structures of the closely related mu-agatoxin-IV (mu-Aga-IV) which were isolated from venom of the American funnel web spider, Agelenopsis aperta. These toxins, which modify the kinetics of neuronal voltage-activated sodium channels in insects, are C-terminally amidated peptides composed to 36 amino acids, including four internal disulfide bonds. The structure of mu-Aga-I was determined by NMR and distance geometry/molecular dynamics calculations. Structural calculations were carried out using 256 interresidue NOE-derived distance restraints and 25 angle restraints obtained from vicinal coupling constants. The peptide contains eight cysteines involved in disulfide bonds, the pairings of which were uncertain and had to be determined from preliminary structure calculations. The toxin has an average rmsd of 0.89 A for the backbone atoms among 38 converged conformers. The structure consists of a well-defined triple-stranded beta-sheet involving residues 7-9, 20-24, and 30-34 and four tight turns. A homologous peptide, mu-Aga-IV, exhibited two distinct and equally populated conformations in solution, which complicated spectral analysis. Analysis of sequential NOE's confirmed that the conformers arose from cis and trans peptide bonds involving a proline at position 15. Models were developed for both conformers based on the mu-Aga-I structure. Our structural data show that the mu-agatoxins, although specific modifiers of sodium channels, share common secondary and tertiary structural motifs with phylogenetically diverse peptide toxins targeting a variety of channel types. The mu-agatoxins add voltage-sensitive sodium channel activity to a growing list of neurotoxic effects elicited by peptide toxins which share the same global fold yet differ in their animal origin and ion channel selectivity.

Agatoxins↗
PubMed1992

Antagonism of synaptosomal calcium channels by subtypes of omega-agatoxins.

Venom of the funnel web spider Agelenopsis aperta inhibits the binding of 125I-omega-conotoxin GVIA (omega-CgTx) to calcium channels in chick brain synaptosomal membranes. Fractionation of the venom by liquid chromatography shows that this inhibitory activity is associated primarily with a diverse class of peptide toxins called omega-agatoxins (omega-Aga). Using binding inhibition as an assay, we purified and identified the novel, 76-amino acid toxin, omega-Aga-IIIA. Inhibition of 125I-omega-CgTx binding to chick synaptosomal membranes by omega-Aga-IIIA and omega-Aga-IIA is correlated with block of potassium-stimulated 45Ca entry into synaptosomes; omega-Aga-IA neither inhibits 125I-omega-CgTx binding nor 45Ca entry under identical conditions. omega-Aga-IIA and omega-Aga-IIIA are 20-30-fold more potent than omega-CgTx as antagonists of synaptosomal calcium channels. However, whereas omega-CgTx completely blocks 45Ca entry into synaptosomes at saturating concentrations, the omega-agatoxins maximally block only 60-70% of 45Ca entry. Pretreatment of synaptosomes with omega-Aga-IIIA occludes block of 45Ca entry by omega-CgTx. The results indicate that, while the omega-agatoxins bind to the entire population of omega-CgTx-sensitive calcium channels in chick synaptosomal membranes, they exert only a partial block of 45Ca flux. Such block could occur via two distinct mechanisms. Toxin binding may alter the kinetics of a homogeneous population of channels, resulting in lower overall conductance upon depolarization. Alternatively, the omega-agatoxins may bind to two distinct channel subtypes, only one of which is blocked as a result of toxin occupation.

Agatoxins↗