Batrachotoxin

BatrachotoxinDaniel PitcherTroy HatfieldMichael Steudle

Batrachotoxin OverviewToxin Profile: Batrachotoxin

Compound: Steroidal alkaloidSource: Golden dart frogs (and related species) of the Amazon and some passerine birds in New Guinea. It is likely sequestered a toxins synthesized by melyrid beetles in their dietTargets: Cardiotoxin and neurotoxin; binds to sodium channels and irreversibly opens them due to conformation changeLD50: .oo2-.007 mg/kg, its maximum activity is at 37 degrees Celsius, making it very potent to humans

3CompoundSteroidal Alkaloid: An alkaloid that had a perhydro-1, 2-cyclopentanophenanthrene nucleus, giving it structural similarities to a sterol. Sterols, such as cholesterol, are nonpolar on one end and polar on the otherAlkaloids are natural occurring compounds that contain mostly nitrogen atoms and are usually basic moleculesSteroidal Alkaloid have similar properties to both these compounds. 4Source: Primary consumerMelyrid BeetlesFound in both frog and bird diets Contain many derivatives of the steroidal skeleton of the batrachotoxinMelyrid beetles dont synthesis steroidal skeletons Beetles consume the molecular skeleton in the form of phytosterols from plants and modify it into batrachotoxin

One beetle species part of the melyrid family; the family all contain Batrachotoxin and other similar steroid alkaloids5Sources: Secondary Consumers

Hooded pitohui Pitohui dichrous

One of the four Pitouhi species of passerine birds found to have sequestered Batrachotoxin in their tissues.

Golden Dart FrogPhyllobates terribilis

The poison dart frog with the highest concentrations of batrachotoxin in the poison glands

http://archive.audubonmagazine.org/webexclusives/plumesOfPoison.html

6Golden Dart Frog Sequestering of BatrachotoxinSequestered in glands spread throughout the body: predominantly dermal glands, but can be found in liver and skeletal muscleMust have developed resistance to batrachotoxin by alteration of the batrachotoxin binding site on Na+ voltage gated ion channels The actual mechanism of uptake (capture, transport, and accumulation in glands) is unknown.

Cross section of frog skin. The large pink glands are dermal poison glands where the toxins are sequestered. 7Bird Sequestering of BatrachotoxinConsumption of Melyrid beetles leads to absorption of the batrachotoxin High concentrations of toxin in dermal tissue like feathers, and skin Smaller concentrations of the toxin found in internal organs (heart, skeletal muscle, liver) Because the toxin is found in internal organs, the bird must not be susceptible to the toxin (indicating a change in amino acid sequence of the binding site).

The white blotch, labeled LD, is a lipid droplet in the skin of a Pitohui species dermal cross section. Lipid droplets are common in bird dermal layers, and it is hypothesized Batrachotoxin is sequestered in the lipid dropletsWhy sequester Batrachotoxin?Reduced predation: the sequestering of toxins in the dermal tissues is a defensive strategy

Increased food opportunities: able to incorporate insects with poisonous compounds like batrachotoxin and other steroidal alkaloids by developing resistance and sequestering themAposematic Coloration of Poison Dart Frogs: Evidence for Anti-predatory Purpose of the Toxins

Four Species of bird and their average toxin concentrations for various tissues

In general, the highest concentrations are in the dermal tissues (feathers and skin) indicating in these birds, batrachotoxin is sequestered in external tissues as a defensive to adaptation to predation

Modes of AbsorptionGI system: frogs and birds consume beetles with the Batrachotoxin Cutaneous: can be absorbed cutaneously by the predators of the frogs (or even unlucky passer-byes who happen to touch areas the frogs have been). GI system: predators of the frogs and birds ingesting themIV/IM: utilized by indigenous hunters in Columbia

Anthropology of BatrachotoxinHow Dart Frogs Got Their NameEmbera Indians of Western Columbia use at least three species of poison dart frogs for their poison dartsUsed for hunting spider monkeys; but can take down animals as large as jaguars using Batrachotoxin and other indigenous plant poisonsFrog most associated with the tribe is the Golden Dart Frog

Unknown Image Source14Anthropology of BatrachotoxinThe tips are coated during the hunt; indigenous peoples carry Golden Dart Frogs with them in wicker baskets

The tips are pressed and rolled on the frogs stimulating secretion of the poison from compression of the skin

15Watch this Video of an Authentic Hunt Using Poison Darts: Its pretty coolhttps://www.youtube.com/watch?v=zd4jDBe9s8g

Synthetic Batrachotoxin A mechanism of a full synthesis Batrachotoxin is yet to be developed. The steroid backbone has been synthesized in a recent 2015 attempt Variations in Batrachotoxin could have medical significance Mechanism of ActionNa+ Voltage Gated Ion ChannelsBatrachotoxin works by binding to the Ion Channel and Causing a conformational Change

Na+ channels remain open, constantly triggering action potentials

Structure of BatrachotoxinBatrachotoxin is a rigid steroidal alkaloid

Contains an oxygen triad and is relatively hydrophobicThis is why it targets the Na+ Channels, it is unable to easily get into the cell

Batrachotoxin s Modification of Na+ ChannelsModifies the Channels in 5 Ways:Shifts the Threshold for Action Potential Generation closer towards depolarization by 30-50 mVGets rid of the fast inactivation (Inactivation gate) of Na+ ChannelsGets rid of the slow inactivation of the Na+ ChannelsReduces Single Channel ConductanceAlters the ion selectivity of the Na+ Channel

All of these actions appear to be irreversible once completedIf the threshold is shifted towards depolarization, an action potential is generated fast initially

Not only do the channels remain open because of Batrachotoxin binding, but action potentials after the channels are bound by batrachotoxin are generated less because the membrane never has the ability to repolarize.

Shifts the Threshold for Action Potential Generation closer towards depolarization by 30-50 mV

Gets rid of the fast inactivation (Inactivation gate) of Na+ ChannelsThe Binding of Batrachotoxin causes the inactivation gate of the voltage gated Na+ ion channel to remain open

Causes the inability of the neuron to generate further action potentials which leads to paralysis.

Gets rid of the slow inactivation of the Na+ ChannelsNormally, the activation gate of the voltage-gated Na+ ion channel would close over time.

The binding of Batrachotoxin prevents that closing and the ion channel remains open irreversibly.

Reduces Single Channel ConductanceSingle Channel Conductance is a measure of the amount of ion (in this case Na+) that flows through a single channel over a given time.

The decrease in the channel conductance when Batrachotoxin is bound leads to the further lessening of the strength of action potentials generated.Alters the ion selectivity of the Na+ ChannelIt is hypothesized that the specific site where Batrachotoxin binds causes the channel to increase the selectivity filter for ions in general.

This increased selectivity feature allows larger ions to pass through the once very selective channel, including NH4+ and Tl+ which could be potentially harmful to the affected neuron.Medical and ResearchApplicationsPoison dart frog researchers noticed that handling the frogs made their skin numb.

Consequently, Batrachotoxin is being investigated for use in topical ointments for skin irritation and pain.

Medical ApplicationsBatrachotoxins potency and selectivity make it a good candidate for neurological drugs.

The toxin and its derivatives/analogs have been patented for future use in preventing pain, relaxing muscles, and reducing pathological neural activity. Medical Applications

The current problem that applies to all current medical applications is the irreversibility of the binding of Batrachotoxin to the Na+ channels.

If a synthetic Batrachotoxin could be created that is able to be reversed, these applications would be more likely to be utilized.Medical Applications

Research ApplicationsThe structure and mechanism of voltage-gated sodium channels have been established, in part, due to research using batrachotoxin.

Batrachotoxins distinctive action on sodium channels make it useful for studies on other toxins/drugs that affect the channels.

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Li, H., Hadid, D., & Ragsdale, D. (2002). The batrachotoxin receptor on the voltage gated sodium channels is guarded by the channel activation gate. Molecular Pharmacology, 61(4), 905-912.Information SourcesMyers, Charles W, John W. Daly, and Borys Malkin. A Dangerously Toxic New Frog (phyllobates) Used by Ember Indians of Western Colombia, with Discussion of Blowgun Fabrication and Dart Poisoning. New York: American Museum of Natural History, 1978.

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Sultan, A, A.R Raza, and K.M Khan. "Towards the Synthesis of Batrachotoxin-Formation of Alkynyl Stannanes." Journal of the Chemical Society of Pakistan. 35.5 (2013): 1371-1385.

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