ω-agatoxin-IVA

Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of ‘P-type’ calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.

Mintz I.M., et al. (1992) P-type calcium channels blocked by the spider toxin omega-Aga-IVA. Nature . PMID: 1311418

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

Bourinet E, et al. (1999) Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat Neurosci. PMID: 10321243

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.

Adams ME. (2004) Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta. Toxicon. PMID: 15066410

Voltage-gated calcium channels are key sources of calcium entry into the cytosol of many excitable tissues. A number of different types of calcium channels have been identified and shown to mediate specialized cellular functions. Because of their fundamental nature, they are important targets for therapeutic intervention in disorders such as hypertension, pain, stroke, and epilepsy. Calcium channel antagonists fall into one of the following three groups: small inorganic ions, large peptide blockers, and small organic molecules. Inorganic ions nonselectively inhibit calcium entry by physical pore occlusion and are of little therapeutic value. Calcium-channel-blocking peptides isolated from various predatory animals such as spiders and cone snails are often highly selective blockers of individual types of calcium channels, either by preventing calcium flux through the pore or by antagonizing channel activation. There are many structure-activity-relation classes of small organic molecules that interact with various sites on the calcium channel protein, with actions ranging from selective high affinity block to relatively nondiscriminatory action on multiple calcium channel isoforms. Detailed interactions with the calcium channel protein are well understood for the dihydropyridine and phenylalkylamine drug classes, whereas we are only beginning to understand the molecular actions of some of the more recently discovered calcium channel blockers. Here, we provide a comprehensive review of pharmacology of high voltage-activated calcium channels.

Doering CJ, Zamponi GW. (2003) Molecular pharmacology of high voltage-activated calcium channels. J Bioenerg Biomembr. PMID: 15000518

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.

Winterfield JR, Swartz KJ. (2000) A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels. J Gen Physiol. PMID: 11055992

Insects have a much smaller repertoire of voltage-gated calcium (Ca(V)) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate Ca(V)1, Ca(V)2, and Ca(V)3 subtypes, although its basal inventory is expanded by alternative splicing and editing of Ca(V) channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect Ca(V) channels, since severe loss-of-function mutations in genes encoding the pore-forming alpha1 subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect Ca(V) channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect Ca(V) channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect Ca(V) channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect Ca(V) channels. This review focuses on peptidic spider toxins that specifically target insect Ca(V) channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests.

King GF. (2007) Modulation of insect Ca(v) channels by peptidic spider toxins. Toxicon. PMID: 17197008

omega-toxins specifically block certain Ca2+ channels in mammalian neurons as well as in dorsal unpaired median neurons isolated from the cockroach Periplaneta americana. In these cockroach neurons both the P/Q-type blockers omega-agatoxin IVA and omega-conotoxin MVIIC but not the N-type Ca2+ channel blocker omega-conotoxin GVIA affected fast Na+ currents sensitive to tetrodotoxin and veratridine. Both omega-toxins enhanced Na+ current decay and thus decreased the amplitudes of the peak currents. They also led to a slower recovery from inactivation. Toxin effects developing within a few min were ot removed upon washing. They were not use-dependent. The description of the effect of omega-conotoxin MVIIC on current kinetics in terms of the Hodgkin-Huxley model revealed that steady-state parameters were not affected whereas the time constant of inactivation was considerably reduced. Under control conditions, the inactivation time constant is similar to the time constant of recovery from inactivation. The toxin-induced increase of the latter time constant and the decrease of the inactivation time constant indicate that inactivation can no longer be described by first-order kinetics.

Wicher D, Penzlin H. (1998) omega-Toxins affect Na+ currents in neurosecretory insect neurons. Receptors Channels. PMID: 9826912

Ca2+ currents in dorsal unpaired median (DUM) neurons isolated from the fifth abdominal ganglion of the cockroach Periplaneta americana were investigated with the whole cell patch-clamp technique. On the basis of kinetic and pharmacological properties, two different Ca2+ currents were separated in these cells: mid/low-voltage-activated (M-LVA) currents and high-voltage-activated (HVA) currents. M-LVA currents had an activation threshold of -50 mV and reached maximal peak values at -10 mV. They were sensitive to depolarized holding potentials and decayed very rapidly. The decay was largely Ca2+ dependent. M-LVA currents were effectively blocked by Cd2+ median inhibiting concentration (IC50 = 9 microM), but they also had a remarkable sensitivity to Ni2+ (IC50 = 19 microM). M-LVA currents were insensitive to vertebrate LVA channel blockers like flunarizine and amiloride. The currents were, however, potently blocked by omega-conotoxin MVIIC (1 microM) and omega-agatoxin IVA (50 nM). The blocking effects of omega-toxins developed fast (time constant tau = 15 s) and were fully reversible after wash. HVA currents activated positive to -30 mV and showed maximal peak currents at + 10 mV. They were resistant to depolarized holding potentials up to -50 mV and decayed in a less pronounced manner than M-LVA currents. HVA currents were potently blocked by Cd2+ (IC50 = 5 microM) but less affected by Ni2+ (IC50 = 40 microM). These currents were reduced by phenylalkylamines like verapamil (10 microM) and benzothiazepines like diltiazem (10 microM), but they were insensitive to dihydropyridines like nifedipine (10 microM) and BAY K 8644 (10 microM). Furthermore, HVA currents were sensitive to omega-conotoxin GVIA (1 microM). The toxin-induced reduction of currents appeared slowly (tau approximately 120 s) and the recovery after wash was incomplete in most cases. The dihydropyridine insensitivity of the phenylalkylamine-sensitive HVA currents is a property the cockroach DUM cells share with other invertebrate neurons. Compared with Ca2+ currents in vertebrates, the DUM neuron current differ considerably from the presently known types. Although there are some similarities concerning kinetics, the pharmacological profile of the cockroach Ca2+ currents especially is very different from profiles already described for vertebrate currents.

Wicher D, Penzlin H. (1997) Ca2+ currents in central insect neurons: electrophysiological and pharmacological properties. J Neurophysiol. PMID: 9120560